BIO1010 Cellular and Biochemical Basis of Life Past Paper PDF

Alessio Caruana BIO1010 Cellular and Biochemical Basis of Life – 16th October 2023 UNIT INTRODUCTION – TECHNIQUES USED IN CELL BIOLOGY Suggested texts: Molecular Biology of the Cell, Alberts, B., Johnson, A., Lewis Miller-Urey Prebiotic Experiment: In 1959 Stanley Miller and Harold Urey tried to generate biochemicals using resources thought to be prevalent under primitive earth conditions: water, methane, ammonia and hydrogen, plus electric arc to simulate lightning – described as PREBIOTIC EXPERIMENTS. Within a week of continuous reflux, more than 20 amino acids were formed as racemic mixtures. Biochemicals of life: - Abundant evidence of major volcanic eruptions 4 billion years ago, which would have released carbon dioxide, nitrogen, hydrogen sulfide, and sulfur dioxide into the atmosphere - In 2007, scientists re-examining sealed vials from original Miller-Urey experiments discovered more than 20 different amino acids produced – More than the 20 that occur naturally - Newer experiments have produced even more diverse molecules - Prebiotic experiments continue to produce racemic mixtures of simple to complex compounds under varying conditions. Stereoisomerism: - Asymmetry such that the two images cannot be superimposed since they are mirror images - These are called enantiomers (D and L isomers) Alessio Caruana How nature selected the L-forrm Amino Acids (2nd June 2006, NS Magazine issue 2554): - Curiously, almost every living organism on Earth uses left-handed amino acids instead of their right handed counterparts. - When made from scratch in the lab, left handed (L) and right handed (D) mirror image forms are equally likely to appear, but in nature, L amino acids dominate - The answer may lie in the way the substances behave as they dissolve in water - Donna Blackmond at Imperial College London and her colleagues dissolved a mixture of solid L and D versions of the amino acid serine in water - They found that a small difference in the initial proportion of one version gets amplified in the resulting solution - So a 100:1 mixture of L- and D-serine produces a solution made up of almost entirely L- serine, but so does a 100:99 mixture - “It doesn’t matter what proportions of solid amino acids you throw in, you always get exactly the same proportions in solution,” says Blackmond. Asteroids and L-forms of amino acids: - Watery asteroids may explain why life is left handed (17th March 2009 by Hazel Muir) - Research suggests that water on asteroids amplified the left-handed amino acid molecule - In the 1990s, scientists found that meteorites contain up to 15% more of the left version too. - “Meteorites would have seeded the Earth with some of the prebiotic compounds like amino acids that are needed to get life started, and also biased the origin of life to the left-handed amino acid form” - Some evidence that polarised starlight preferentially destroyer right handed amino acids on asteroids - Previous experiments have shown that water amplified the asymmetry - They studied an amino acid called isovaline in six meteorites that showed evidence of ancient exposure to liquid water for about 1000-10,000 years - The longer water persisted in the rock, the stronger its left-handed isovaline bias, the team found Cellular and Biochemical Basis of Life: - But are they all cells? - The cellular basis of life – the prokaryotic and the eukaryotic cell, viruses - Small but complex structures - Lots of intercellular differences - Sometimes hard to see and understand structure - Molecular composition is very complex - Oftentimes very hard to understand how these molecules interact – omics making things a bit clearer (In biology the word omics refers to the sum of constituents within a cell. The omics sciences share the overarching aim of identifying, describing, and quantifying the biomolecules and molecular processes that contribute to the form and function of cells and tissues. – www.brittanica.com) Alessio Caruana Viruses: - While viruses are generally very small, even when compared to cells, recently giant viruses have been found in sludge, over twice the size of regular viruses, over 1 micrometre. These have around 2500 genes Bacteriophage: - [insert good diagram] Viroids: - Plant pathogens - Have short RNA (200-300 nucleobase stretch) - Mostly circular, single stranded RNA but have some double stranded regions – highly complementary regions - Do not encode for any protein - Replication by rolling circle mechanism involving RNA Polymerase II - No protein coat - May hold clues to early origin of life - These can have significant effect on plants, including distorted or stunted growth Prions – Protein infectious particles - Misfolded, protease resistant proteins which can mediate transmission of disease - Involved in a number of fatal sheep, cattle and human encephalopathies – brain degeneration diseases - In humans these include – Alzheimer’s, Creutzfeldt-Jakob, fatal familial insomnia, Kuru - Consist of a single infectious sialoglycoprotein called PrP - Induces spontaneous alpha to beta transformation of peptide which aggregates into amyloid fibrils – Kills neurons - Do not contain any nucleic acids Cell Biology tools: - Huge range of tools developed over the past 50 years - Tools determine the investigation’s outcome - New discoveries often follow: o New experimental tools o Novel experimental methods using existing tools - Electron microscopy revolutionised cell biology o Better understanding of structures o Structural organisation of cells - Other advanced tools: Alessio Caruana o Confocal florescence microscopy, atomic force microscopy, flow cytometry, etc. o DNA analysis tools, PCR Experimental Methods in Cell Biology: - Different types of microscopy techniques - Animal cell culture - Cell fractionation and centrifugation - Methods involving analysis of biomolecules - Use of isotopes as tracers - X-ray diffraction and other methods of investigating macromolecules Experimental Methods: - Microscopy 1: Light Microscopy, Phase Contract, Fluorescent Microscopy - Microscopy 2: Confocal, TEM, SEM, STEM, Atomic Force - Cell culture Techniques and Xenopus laevis as source of large quantities of oocytes - Staining techniques, specimen preparation and immunohistochemistry - Cell fractionation techniques – isolation of sub cellular organelles/components o Preparative o Density gradient centrifugation - Flow cytometry - Separation Techniques for large biomolecules – chromatography, electrophoresis - Separation and identification of Proteins – Western Blots - Separation of DNA or its fragments – Polyacrylamide gel electrophoresis - RNA nucleic acid separation/hybridisation – Northern Blots - DNA separation/hybridisation – Southern Blots 23rd October 2023 – MICROSCOPY 1: LIGHT MICROSCOPY, PHASE CONTRAST, DARK FIELD, FLUORESCENT These lectures will review: - Different types of microscopes – light, electron, other specialised microscopes Scale – Macromolecules to atoms - Progression – ribosomes to macromolecules to atoms - Details of proteins and toms are only now just coming within the resolving power of the electron microscope (0.7 angstrom units) - Units of length commonly employed in microscopy o Micrometre – 10-6m o Nanometre – 10-9m o Angstrom unit – 10-10m o Refer to the most powerful microscope – FEI Titan 2 EM Alessio Caruana Log scale: - Importance of understanding logarithmic scale Comparing different microscopic techniques: - X-ray crystallography: o Sample must be crystallised in a lattice structure o Any size molecule o Atomic resolution but crystallisation may take years and damage protein structure - Nuclear magnetic resonance: o Sample must be dissolved in water o Small molecules o Closer to real protein structure but larger proteins cannot be resolved - Cryo-Electron Microscopy o Sample is frozen in its native state o Larger molecules o Near-atomic resolution, fast sample preparation Light microscope image: - Phase contrast gives very crisp light microscope images - Uses no stains but still creates contrast - Harnesses changes in waves amplitude and phase Conventional Light Microscope: - Light path in compound microscope: - Lenses in condenser focus light on the specimen - Objective and eyepiece lenses arranged to focus image of specimen in the eye - Lens manufacture important to eliminate defects – aberrations (two types) Resolving Power (R) - Resolving power – ability of a lens system to distinguish between adjacent points as separate objects - R is the ability to form istinct images of two points with close angular separation Alessio Caruana - Limit of resolution – minimum distance at which two point can be seen as two distinct objects - The resolving power depends on: o Wavelength of light o Numerical aperture (NA) of the lens - NA = n0 sin θ0max (alpha may be used instead of theta) o Lens defects or aberrations Resolving power and wavelength: - To distinguish between two closely situated points the shorter the wavelength, the better - Thus blue light gives a better resolution than red light - R of a light microscope with white light is 2 micrometres - R for violet light is about 1.87 micrometres - R for an electron microscope is about 5nm, because electrons have a much lower wavelength than visible light R and numerical aperture (NA): - NA is a function of the cone of light passing through the lens – its light collecting ability - Width of light cone collected by objective lens depends on the Angular aperture (A) - NA is calculated as n sin α where: o n is the refractive index o α is half the angular aperture (A) - NA cannot be more than 1 for dry lenses but as high as 1.4 for oil immersion lenses - Advantages: o The higher the NA, the greater the resolution and the brighter the image - Disadvantages: o The higher the NA, the shorter the working distance o The higher the NA, the smaller the depth of field Calculating resolving power: - Limitations of the values alpha, lambda and n o Alpha for the best lens is about 70o o Shortest wavelength of visible light is blue (450nm) o Highest resolution lenses – oil immersion (n = 1.56) - Theoretical resolution limit of a light microscope using visible light – about 200nm using blue or violet light (450-400nm) and a NA of 1.4664 when using oil immersion Alessio Caruana Limitations to resolving power: - Resolving power is additionally limiting by a number of factors: o Interference between waves of different phases – affects image brightness o Diffraction effects since the lens aperture acts as a tiny diffraction hole giving rise to an edge effect and blurring of the image Interference and diffraction: - When light waves combine in phase, the resultant wave has a larger amplitude and increased brightness - Light waves partly cancel when out of phase – Produce a wave with decreased amplitude and decreased brightness - Edge effect – the diffraction effects observed at high magnification when light passes the edges of a solid object placed between the light source and the observer Lens aberrations: - Spherical aberrations arise due to lens curvature – not all the parts of an object are focused in one plane - Chromatic aberration image has coloured fringes – lens acts as a prism and diffracts light into different wavelengths - Thus achromatic lenses and plan-achromatic lenses correct for these defects Limitations of light microscopy: - Optical thickness of specimen – how depth of material/medium affects image appearance - Intensity of light (or other radiation) at known frequency is reduced by a given factor - Optical thickness of specimen = o Difference of refractive indices of object/specimen and medium – a measure of contrast o X is the thickness of specimen parallel to the direction of light - The larger the optical thickness, the poorer the image appearance Increasing contrast – STAINS: - Stains help reduce the amplitude of particular wavelengths passing through them, thus a coloured image of the cell is obtained - However stains also distort/damage tissues and introduce artifacts - Light through an unstained living cell undergoes little amplitude change, structural details cannot be seen even if highly magnified o Phase of the light is altered by its passage through different thickness of the cell o Small phase differences made visible by exploiting interference effects using a phase- contrast or a differential-interference contrast microscope Modifications of the light microscope: - Bright field microscopy and phase contract microscopy o Bright field microscopy – specimen seen against bright background o Phase contract microscopy – does away with staining, exploits differences in the thickness of different cell parts ▪ Produces a small phase shift relative to thinner/less dense regions Alessio Caruana ▪ Small phase shifts are converted into amplitude or contrast changes in the image ▪ Characterised by a bright halo around the edge of the live cell - Differential interference and dark field microscopy o Nomarski differential-interference-contrast microscopy makes use of computer image processing by electronically subtracting background image irregularities o Dark-field microscopy – specimen illuminated from the side ▪ Particles or structures act as light stoppers and scatter light ▪ Objects shine and their outline is seen against a black background Light microscopy – steps in tissue preparation: 1. Fixing 2. Embedding and sectioning 3. Dewaxing 4. Staining 5. Dehydration 6. Mounting Fixation: - Termination of life processes and prevention of decay/deterioration - May introduce distortions or artifacts - A variety of methods used – desiccation, heating, 10% formalin, 70% alcohol, and compound fixatives - There are other compound fixatives such as Zenker’s, Formol-saline and Carnoy’s Embedding and sectioning - Preparation for embedding usually involves dehydration and clearing - A fixed specimen is embedded-impregnated with wax of resin - Specimen is sectioned with a microtome - A ribbon of thin wax sections are carefully collected on slide or water - Dewaxing (3.) refers to the removal of wax by solvents (xylene) prior to staining. Alessio Caruana Staining - Stains contain a chromophore that may be basic, acidic or neutral - Stains usually require mordants or accentuators to bring out colour - Progressive, regressive, counter and double staining techniques can be used - A counterstain I a stain with colour contrasting to the principal stain, makes the stained structure more easily visible - E.g. – H & E counterstaining of urine-collecting ducts of the kidney and Gram stain anthrax Haematoxylin and Eosin: - Designed to show: o Basophilic structures (nuclei) blue, black, purple or grey o Acidophilic structures (extracellular and cytoplasmic proteins) in shades of pink and red - Haematoxylin is a basic blue chemical, develops colouring properties on oxidation to haematin - Haematin needs a mordant (metal salt) that brings contact between dye and tissue Eosin - Eosin is an acidic red-pink dye, that shows up in basic parts of the cell - Can be used to stain cytoplasm, collagen and muscle fibres - In solution – dye molecule is negatively charged, attaches to positive site in the tissue by salt bridges Haematoxylin and Eosin – Common Cell Stain - Haematoxylin staining is shown as blue/purple colour that stains acidic cell contents – nuclear chromatin - Eosin is a red-pink dye that stains basic or neutral cell contents – cytoplasm Fast green and safranin - Fast green used to stain cellulose green - Safranin stains lignified walls (xylem vessels) red. More staining - Intestinal wall – Mallory’s trichrome stain: blue for collagen, pink/purple for cytoplasm - Epithelial cells with bright red secretory granules (eosinophilic). Nuclei are dark blue/purple stained with haematoxylin The thymus in H & E - Stains predominantly blue, large quantity of small nuclei (acidic nucleic acids) attract haematoxylin - The thymus is packed with lymphocytes with haematoxylin darkly stained purple nuclei - Centre vessel and cytoplasm stains red with eosin Principles of fluorescent staining - Fluorescence occurs when: o A fluorophore is excited by light of wavelength 𝜆𝑥 Alessio Caruana o Its electrons are raised from ground state to an excited state o On its return to ground state, the fluorophore emits light of a longer 𝜆 o This is usually in the visible spectrum and hence of a lower energy content o This can be separated from the incident light using appropriate optical filters Excitation and emission wavelengths - Maximum excitation and emission wavelengths of fluorescent dyes - The photon emitted by a dye molecule is of lower energy (longer wavelength) than the photon absorbed Fluorescent microscope optics: Fluorescent microscope features - A filter set consists of two barrier filters (1 & 3) and a dichroic (beam-splitting) mirror (2) - In this example, the filter set for detection of the fluorescent molecule fluorescein is shown - A source of white or UV light - An excitation filter (1) – removes most of the wavelengths present in the white light except the one that will excite the fluorescent dye - Fluorescent light is usually quite weak and therefore similar wavelengths must be reduced or completely eliminated - Reflecting surfaces required to deflect the excitation beam are usually prisms made of quartz especially if UV is used - The excitation beam is passed through the fluorescent dye stained object - The out-coming light will be fluorescent but with it there is some excitation light as well - A barrier filter is the final step before the fluorescent light enters the microscope objective Alessio Caruana - The last barrier filters out light of excitation so that only light of emission is seen Natural fluorescent compounds - Some compounds are naturally fluorescent on exposure to certain wavelengths of light – chlorophyll, sesquiterpene lactones and colchicine Fluorescent compounds - Most fluorescent compounds are synthetic inorganic or organic compounds - Common everyday use in detergents to impart a fluorescent white colour to fabric - Use of fluorescent dyes in biology is mostly to highlight certain features in microscopy as certain dyes combine directly with particular compounds - E.g. Acridine orange - A number of fluorophores are used to label cell components directly as for example nucleic acids - Fluorophores can also be combined with antibodies for a particular antigenic molecule (protein, carbohydrates, lipids) & can act as tracers to locate tat molecule of interest within the cell or in a tissue - Fluorescent dyes usually emit a bright coloured light that gradually decreases with exposure to other light sources – quenching which is the equivalent of bleaching of the fluorophore Example of fluorescent stains no. 1 - Acridine orange and quinacrine dihydrochloride – acridine dyes o Intercalates between bases of double stranded RNA and DNA or by external ionic binding and stains yellowish green o Stacks on charged phosphate groups on single stranded nucleic acids (example mRNA) where it fluoresces red - For eukaryotic cells, simultaneously make a DNA measurement (double-stranded - green fluorescence) and an RNA measurement (single-stranded – red fluorescence) Nuclear fluorescent stains - Most bind stoichiometrically to nucleic acids (in an amount proportional to the quantity of DNA present) e.g. Propidium iodide, ethidium bromide, quinacrine dihydrochloride, etc. - Can be used for quantitative measurements flow-cytometry – cell ploidy and cell cycle visualisation is undertaken - Changes in fluorescent properties of the fluorophore before and after binding to the nucleic acids - Most do not pass freely across the cell membrane – cells must be fixed and/or permeabilised before staining - Exception is benzimides (H-33342 and H-33258) – penetrate cell membrane without permeabilization Examples of fluorescent stains no. 2 - Propidium iodide and ethidium bromide o Intercalate between the purine and pyrimidine bases in double stranded nucleic acids o Bind to both RNA (double stranded) and DNA o The former has to be removed if only DNA is to be measured Alessio Caruana o They are excited by UV or blue light to give red or orange fluorescence More fluorescent stains no. 3 - Benzimides Hoechst 33342 and Hoechst 33258 stains – these bind to adenine-thymidine (AT) rich regions being specific to thymidine - Fluoresce blue when excited by UV - If the concentration of bound dye increase above a certain critical value, fluorescence shifts from blue to red - Since it is transported freely in and out of the cell, fluorescence depends heavily on the conditions of incubation More fluorescent stains no. 4 - Fluorescein isothiocyanate (FITC) is the fluorophore of choice in immunohistochemistry. It is usually bound to antibodies or a conjugate and fluoresces green when excited. Typically it is conjugated with: o Phalloidin – a compound extracted from the poisonous mushroom Amanita phalloides that binds the cytoskeletal elements o Annexin V Direct and indirect immunocytochemistry - Immunocytochemistry can be direct and indirect - Both involve marker-linked antibodies - Major drawbacks for direct immunostaining - How are these avoided in indirect immunostaining Indirect immunocytochemistry - Very sensitive detection method - Primary antibody is itself recognised by many molecules of the secondary antibody - Secondary antibody is covalently coupled to a marker molecule – makes it readily detectable – include fluorescent dyes, horseradish peroxidase, colloidal gold spheres, enzymes alkaline phosphatase or peroxidase Multi-fluorescent Probe Microscopy - Three fluorescent probes have been used to stain three different cellular components of cell in mitosis o The spindle microtubules are revealed with a green fluorescent antibody Alessio Caruana o Centromeres with a red fluorescent antibody o The DNA of the condensed chromosomes with the blue fluorescent dye DAPI Indirect immunocytochemistry – 6th November 2023 - Advantages: o Microtubules distribution using TEM of cultured epithelial cell o Same area stained with fluorescent antibodies against tubulin using indirect immunocytochemistry Annexin V – FITC staining - During apoptosis, phosphatidylserine (PS), is translocated to the outer leaflet of plasma membrane using scramblase – bidirectional (flippases – from exoplasmic to cytosotic and floppases – reverse) - PS serves as an apoptosis marker for macrophage recognition and engulfment - PS-binding protein – Annexin V is a Ca2+ dependent phospholipid-binding protein - Can bind other phospholipids, but highest affinity for PS - Annexin V can be conjugated to fluorescein isothiocyanate (FITC) – utilised as a probe for the quantification of apoptotic cells. Apoptosis vs. Necrosis - Stages of cell death: o Apoptotic cell – early stage with PS transferred to outside, membrane still intact – no PI staining o Necrotic cell, with some PS externalised, however membrane structure compromised – PI stain accumulating in cell FISH – Fluorescent in situ hybridisation - Detects, localises specific DNA sequence on chromosomes - Prepare small probe – getting a purified section of genome specific to target gene by nick translation - Nick Translation – tagging technique using DNase and digoxigenin or biotin to tag probe - Denature chromatin (formamide at 42C) and add tagged probe - Probe binds to that part of the chromosome with which it shows a high degree of sequence complementarity - Seek out probe with antibody (against what?) tagged to fluorophore Alessio Caruana - Nick translation is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labelled analogues. Avidin is a tetrameric biotin-binding protein. Significance of Objective Lens Data - x10 o x10 objective lens magnification - 4.0/0.65 o 4.0 plan and type of correction for respective tube length o ).65 numerical aperture (NA) - 160/0.17 o 160mm is tube length between objective and ocular lens o 0.17mm coverslip thickness MICROSCOPY 2 Advanced Microscopy Confocal scanning Fluorsecent Microscopy (CFSM) - Fine laser beam – scans through several layers in a cell or tissue - Virtually cuts sections of variable thickness - Reveals differences in structure at various levels - Builds a 3D image of the cell contents Alessio Caruana - Stained with fluorescent dyes – structures easily identified - - E.g. Phalloidin FITC for the actin cytoskeleton and with Hoechst stain for the DNA/RNA contents of the nucleus - E.g. Rhodamine B – a protein specific dye (stains red) How the CSFM works - A beam of laser light of the correct excitation frequency is projected through a pin hole onto the stained specimen - The stained specimen emits the fluorescence - The incident and fluorescent light are reflected onto a detector - However only a small point of light from the object is allowed at a time thus building a three dimensional image this way - Involves complex computational power Confocal microscope optics - Laser illuminates a small pinhole on plate A - Image focused at a single point in the 3D specimen - Emitted fluorescence from this focal point focused at a second (confocal) pinhole on plate B - Emitted light from elsewhere in the specimen is not focused – does not contribute to the final image (C) - Beam of light scanned across specimen, a very sharp 2D image of the exact plane of focus is built up Alessio Caruana Conventional vs. confocal - Conventional, unprocessed image – blurred by fluorescent structures above and below the plane of focus - In the confocal image, this out-of-focus information is removed – gives a crisp optical image of the cells in the embryo. 3D reconstruction from CSFM images - CSFM images at different depths through the specimen recombined to give a 3D view Electron Microscopy (EM) - The lenses in LM made of glass or quartz - The lenses in the EM are magnetic coils - LM uses light with an average wavelength of 0.53 micrometres (530 nanometres) - EM uses an electron beam – average wavelength of 0.004 nm at an accelerating voltage of 100kV - The electron microscope required that the specimen be placed in a vacuum – dead, ultrathin, specially stained Limit of resolution for EM - Theoretical resolving power should be 0.002nm but in actual fact it is around 0.1nm - TEM of a thin layer of gold shows: o The individual files of atoms in the crystal Alessio Caruana o The distance between adjacent files of gold atoms is about 0.2 nm Common EM Fixative and stain - Two reactive aldehyde groups of glutaraldehyde – cross-linking of molecules, forms covalent bonds between them - Osmium tetroxide – reduced by many organic compounds forming cross-linked complexes - OsO4 especially useful for fixing cell membranes – reacts with the unsaturated C=C double bonds present in many fatty acids Ultrathin sectioning - For conventional TEM it is usual to examine section of 50-70 nm thick - These thin sections give sharply focused images at high magnification - For high magnification (>x30,000), even thinner sections must be made, provided that there is still sufficient contrast present in the specimen. Preparation of Metal-shadowing: Positive staining vs. negative staining - Stains the specimen or stains the surroundings respectively Immunogold Electron Microscopy staining - Thin section yeast mitotic spindle with microtubules Alessio Caruana - Antibodies against 4 proteins used to localise position within the spindle pole body of yeast - Antibodies linked to colloidal gold particles to allow for detection of the antibody via TEM - Black dots reveal where each protein is located - NB antibodies must be used separately - 4 different images for due to four different proteins with each antibody Scanning electron microscope (SEM) - Scanning beam of electrons focused on specimen by electromagnetic coil lenses - Quantity of electrons scattered or emitted from surface of the specimen measured by detector - An image is built up on a video screen - SEM creates 3D images with great depth of focus and a resolution between 3 nm and 20 nm depending on the instrument Atomic Force Microscope - Precision instrument – fine laser beam directed on a silicon cantilever etched with a fine tipped point - Part of cantilever surface is coated with a layer of gold – acts as a mirror - Cantilever placed in holding strip, fixed in place on a holder equipped with a fine micrometre stage - Stage vibrated by a piezo crystal to maintain Z height - Stage adjusted – laser beam hits the cantilever to give optimum reflection onto a photodiode - Tip brought to surface – different types of forces deflect cantilever - Tip moved in tapping mode or scanning mode - The specimen is fixed to iron stub Alessio Caruana - Cantilever slowly applied to surface at a certain frequency and force load - Typical areas scanned are 1 um2 to 100nm2 and scan rates of 1.5Hz - Tip used to induce defects in a specimen – scan a small area at high speed (122Hz) and high force load - Image built up by integration of reflected signals from photodiode - Use of AFM software to flatten image and make necessary measurements AFM Operation - AFM can be operated in two modes: o Tapping mode – most suitable for biological samples o Scanning mode – most suitable for non-biological samples - If scanning mode is applied for biological samples, the chances are that the sample is swept off the mica surface - Tapping mode AFM is applied to o Liposomes collapsed on freshly cleaved mica surface and hydrated with phosphate buffered saline (PBS) o Membranes to elicit protein pump structures Pulse Chase Autoradiography - Trace pathway of formation of cell products such as proteins or other metabolites - Unravels substrate to product path - Pulse – involves addition of a measured dose of the radioactive tracer which acts for a short time - Chase – addition of excess unlabelled compound - Tracer pulse is localised and followed as it moves through different cell parts Pulse Chase Autoradiography – 13th November 2023 - The chambers labelled A, B, C, D represent: o Either different compartments in the cell (detected by autoradiography or by cell- fractionation experiments) o Or different chemical compounds (detected by chromatography or other chemical methods) - Picture of cell taken at intervals – locate position of radioactive tracers - Tracer/pulse incorporated into cell components e.g. 3H Thymidine (Tritiated thymidine) incorporated into DNA but not RNA (not uracil) - Chased by unlabelled thymidine - Detected by photographic means Alessio Caruana - Other heavy isotopes such as 14C, 32P or 35S can also be used - Labelled tissue of cultured cells on glass slide covered with a glycerine and AgBr photographic emulsion - AgBr decomposes into Ag when treated with light/radioactivity - Unused AgBr fixed from further development - Developed Ag eventually photographed as a dark precipitate - Similar experiments were important to establish intra-cellular pathway for newly synthesised secretory proteins. EM Protein Autoradiography - Pulse-chase experiment – pancreatic cells fed 3H-leucine for 5 minutes (the pulse) followed by excess unlabelled leucine (the chase) - Amino acid is largely incorporated into insulin, which is destined for secretion - After a 10-minute chase, the labelled protein has moved from rough ER to the Golgi stacks (A) where its position is revealed by the black silver grains in the photographic emulsion - After a further 45-minute chase, the labelled protein is found in electron-dense secretory granules (B). Autoradiography - Radiation must not be too penetrating – must affect only a small area in cell - If Ag particles are deposited all over the cell, process will not resolve exact radiolabel site. - Ideally the radioactivity should be strong but over a short distance 3 - H emits beta particles which have a short path in the range of 0.4-0.5 microns. CELL CULTURE TECHNIQUES Why? - Need for CCT to carry out certain studies o Progression of a process with time o Cell cycle changes o Intracellular activity and relation between organelles o DNA replication and transcription o Movement of RNA and protein synthesis o Energy metabolism o Translocation of hormone receptor complexes o Signal transduction processes What is Cell Culturing? - Cells from a certain tissue cultured outside the body - Two types: o Monolayer (adherent) o Suspension culture (non-adherent) - Uses specialised media – balanced salts and supplements - Culture conditions must be highly controlled – temperature, CO2, O2, pH, nutrients, waste products Alessio Caruana Best sources of cells - Foetal or neonatal tissues are the best sources of viable cells – short lived cultures (SLC) - Umbilical cord or cord blood are ideal sources – stem cells/SLC - Haematopoietic tissue – SLC - Tumour tissue is also a good source of viable cells – Long Lived Cultures Step 1 – Isolate cells of a uniform type - Tissues are a mixture of different cells held in extracellular matrix - Cells of explant are dispersed enzymatically o Use Chelating agents – trisodium citrate o Use enzymes – collagenase, trypsin, pronase or dispase - Cells derived from explant are a mixture and need to be sorted: o Centrifugation o Differential cell adhesion o Antibody assisted/fluorescence activated cell sorting Approaches to cell sorting: - Centrifugation – used to separate fragments/debris o Fragments/other debris, small from large cells or dense from light cells - Differential cell adhesion – cells’ variable affinity to adhere to glass or plastic – rapidly adherent to slow adherent - Antibody-assisted cell sorting – specific antibodies raised to bind surface of one cell type o Very specific, high accuracy o Antibody attached to matrix – affinity surface o Matrix of collagen or polysaccharide-coated beads o Recover bound cells – gentle shaking or matrix digestion - Fluorescent-activated cell sorting Fluorescent activated cell sorting - Stream of cells pass across laser beam – drops monitored for fluorescence - Small groups of cells given a charge: o Negative (fluorescent) o Positive (non-fluorescent) - Droplets deflected by electric field according to charge o Cell clumps fall to waste o Cell concentration adjusted so that most droplets contain no cells and flow to a waste container Alessio Caruana Step 2 – Establishing a cell line - Cells are maintained over several generations where cells are repeatedly sub-cultured – continuous cell line develops o Cells from healthy tissue die after a few generations – Primary culture – short lived o Cells from malignant tissue – longer-lived - Better chances of establishing a continuous cell line (CCL) if starting material is o Genetically transformed (e.g. cancerous tissue) o Virally infected and immortalised (SV40 virus, EBV) o Transformed or immortal Adherent and suspension CCL - Adherent Cultures o From adherent and solid tissue o Cells secrete matrix Alessio Caruana - Suspension cultures o From blood forming tissue o No matrix secreted Conditions for Cell culture - A completely sterile environment o Avoid microbial or chemical contamination o All materials, equipment, air must be sterile – use laminar flow cabinet with filter sterile air - Reproduce the physical and nutritional conditions present in an organism, control: o Temperature, oxygen, carbon dioxide o pH and osmolality Cell Culture Medium - Medium supplies nutrients and energy requirements for cell growth and multiplication o Biochemicals or precursors for other biochemicals o Basal medium – BSS, aa, energy source, vitamins, etc. o Buffering system, e.g. NaHCO3 o Accessory factors – trace elements, nucleosides and pyruvate (TCA), growth factors, hormones o Supplement FBS (used at 10-20%) - Most frequently used media are: o Dulbecco’s modified Eagles medium – DMEM o Roswell Park Memorial Institute – RPMI 1640 Xenopus laevis and cell biology - Xenopus laevis – the African Clawed Toad - Extensively used in fertilisation and cancer studies - Advantages of using this species are: o Easy to maintain food and temperature needs o Resistant to disease o Female frog induced to lay eggs by injecting human chorionic gonadotropin (HCG) o Males can also be stimulated to mate o Life cycle from fertilised eggs to maturity 5 x 106 Da Affinity chromatography - Insoluble matrix covalently linked to specific ligand, e.g. antibody/enzyme substrate, binds specific protein - Antigen/enzyme molecules bind to immobilised substrates - Enzyme eluted using cocentrated solution of the free form of the substrate molecule - Antigens that bind to immobilised antibodies can be eluted by dissociating the antibody- antigen complex with: o Concentrated salt solutions o Solutions of high or low pH - High degrees of purification are often achieved in a single pass through an affinity column Protein Column Chromatography – 27th November 2023 - Three different chromatographic steps are used in succession to purify a protein - Cell homogenate first fractionated by allowing it to percolate through an ion-exchange resin packed into a column - The column is washed, and the bound proteins eluted by passing a solution containing a gradually increasing concentration of salt through the column - Proteins with the lowest attraction for the ion-exchange resin pass directly through the column and are collected in the earliest fractions eluted from the bottom of the column Alessio Caruana - The remaining proteins are eluted in sequence according to their attraction for the resin – those proteins binding most tightly to the resin requiring the highest concentration of salt to remove them - The protein of interest is eluted in several fractions and detected by its enzymatic activity - The fractions with the same activity pooled and then applied to a second, gel-filtration column Chromatography sequence Affinity Chromatography and SDS-PAGE - The elution position of the still-impure protein determined by its enzymatic activity - The active fractions pooled and purified on an affinity column that contains an immobilised substrate of the enzyme - Affinity purification of cyclin-binding proteins from S. cerevisiae, as analysed by PAGE o Lane 1 is a total cell extract o Lane 2 shows the proteins eluted from an affinity column containing cyclin B2 o Lane 3 shows one major protein eluted from a cyclin B3 affinity column - Proteins in lanes 2 and 3 eluted with salt and the gels were stained with Coomassie blue Alessio Caruana Alessio Caruana SDS PAGE Electrophoresis - Two chemicals used to solubilise proteins for SDS polyacrylamide – gel electrophoresis SDS PAGE - A detergent and reducing agent - Sodium dodecyl sulfate (SDS) – 12C aliphatic chain detergent - Beta-mercaptoethanol – powerful reducing agent - SDS imparts a negative charge - Mercaptoethanol opens up proteins by destroying stabilising disulfide bridges - Polypeptide chains form (-) SDS-protein complex - Neg. charged SDS-protein complex migrates through polyacrylamide gel pores - NB: if the protein contains a large amount of carbohydrate MW estimated by SDS-PAGE can be misleading. If the protein contains a large amount of carbohydrate, it will move anomalously on the gel and its apparent molecular weight estimated by SDS-PAGE will be misleading. - Speed of migration is greater for smaller polypeptides - Technique to determine the MW of polypeptide chain – use a standard MW marker Alessio Caruana Protein Isoelectric Separation - Protein may naturally carry no charge or a net negative or positive charge - At low pH or high [H+] most proteins have a net positive charge o Carboxylic acid group (-COOH) of proteins is uncharged o Nitrogen-containing basic groups are fully charged (-NH3+) - At high pH or low [H+] most proteins have a net negative charge o Carboxylic acid groups negatively charged (-COO-) o Basic groups tends to be uncharged (-NH2) - At its isoelectric pH – positive and negative charges balance – a protein has no net charge Separation of Protein Molecules by Isoelectric Focusing Isoelectric Focusing - Isoelectric point found o A tube containing a fixed pH gradient o Subjected to a strong electric field in the appropriate direction o Each protein species migrates o Forms a sharp band at its isoelectric pH Two-dimensional PAGE - Total proteins in E. coli bacteria separated on single gel - Each corresponds to a different polypeptide - Proteins first separated by isoelectric focusing from left to right - Further fractionated by MW using SDS-PAGE from top to bottom - Additional notes: o Bacteria fed with labelled amino acids o Therefore all their proteins were radioactive o Detected by autoradiography o Note proteins present in different amounts Alessio Caruana Western blotting - Total proteins from dividing tobacco cell culture separated by 2D-PAGE - Positions revealed by Coomassie blue stain - Separated proteins on an identical gel transferred to nitrocellulose sheet - Sheet exposed to antibody specific for CDK threonine phosphorylated proteins - Immunoblotting – the positions of antibody recognised proteins revealed by an enzyme- linked second antibody Principles of centrifugation - Sedimenting force on particle o SF = Mass x centrifugal field o SF = m r ω2 ▪ m = mass of particle Alessio Caruana ▪ ω = angular velocity of rotor (radians / sec) ▪ r = radius (i.e. distance of particle from axic of rotation Relative Centrifugal force (RCF) Sedimentation Coefficient (S) 𝑉 - S = Rate of movement down tube / Centrifugal force = 𝜔2 𝑋 o Where V is particle velocity o Omega is the angular velocity o X is thee viscosity of medium - S is increased for particles of: o Larger mass o More compact structures of equal particle mass (frictional coefficient is less) - Svedberg is the unit of sedimentation BASIC BIOCHEMICALS – AMINO ACIDS Proteins and amino acids - Proteins – highly complex organic compounds found in all living cells - The most abundant class of all biological molecules - The most functionally diverse molecules in living systems - Every life process depends on these molecules - Huge variety of proteins from enzymes, hormones, O2 and e- carriers, energy converters, motor and skeletal proteins, etc. Important proteins in cells - Regular array of two types of proteins in insect flight muscles – actin and myosin - Proteins can be enzymes - DNA interacts with different types of proteins called histones - These play a part in regulating DNA expression and help in protecting and packaging of DNA into nucleosomes The Organisation of DNA in a chromosome - A nucleosome contains a protein core made of eight histone molecules - The nucleosome core particle is surrounded by two turns of DNA with a linker DNA strand between nucleosomes Amino acids (aa) - What do all proteins have in common? Aa – building blocks of proteins - Over 300 different aa described in biological systems, especially plants - The strange case of β-N-methylamino-l-alanine (BMAA) – a neurotoxic, non-protein aa from cyanobacteria. Brain degenerative disease similar to Parkinsons in GUAM - Only 20 aa commonly found as constituents of proteins - Amino acids released by the digestion of food proteins - Absorbed and carried in the bloodstream to the body ccells Alessio Caruana - Used for growth, maintenance and repair - Cellular catabolism breaks amino acids down into smaller fragments Non-essential and essential aa - Non-essential amino acids are necessary in metabolism but can be synthesised in the human or animal body when needed - Others cannot be synthesised in sufficient quantities and are termed essential – nine aa - They must be provided in the diet Structure of Amino Acids - Any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen and in certain cases sulfur - 20 found as common constituents of mammalian cells - They are the only amino acids coded for by the DNA, the genetic material of the cell The 20 aa found in proteins - Both three letter and one-letter abbreviations listed below Structure of amino acids - Amino acids are characterised by the presence of o An amino group (NH2) o A carboxyl group (COOH) o A distinctive side chain or R group - These two functional groups and R group are attached to the same carbon at the end of the compound described as the α-carbon Groups of an amino acid - Amino and carboxylic acid functional and R groups of an aa - Central alpha carbon - Note that the NH2 is a basic group and the COOH is an acidic group Effect of pH on amino acids - The predominant charged group at low pH is NH3+ - The predominant charged group at high pH is COO- - At physiological pH (7.35-[7.4]-7.45) o The carboxyl group is dissociated forming a negatively charged carboxylate ion o The amino group is protonated - At a result a dipolar internal ion or “Zwitterion” is formed - The ionic groups of opposite charge are in equal numbers Stereoisomerism - Every amino acid except glycine shows optical activity through rotation of plane-polarised light - They can therefore occur as either of two optically active stereoisomers, d (+) and l (-) forms - Isomerism arises when an asymmetric atom is present – four DIFFERENT groups attached - In this case, on α-carbon atom, chirality arises - Asymmetry such that the two images cannot be superimposed - enantiomers Alessio Caruana - Both isomers occur in nature, though the more common isomer is the l-form - The representation of the bond orientation of amino acids follows the Fischer convention o The horizontal (or wedge shaped) bonds protrude out of the plane of the paper o The vertical (or dotted) bonds recede behind the plane of the paper Priority and handedness - By convention the groups are assigned priority by atomic mass - Start with group with largest atomic mass attached to C - Then for groups of equal priority take attached atoms - As one moves from one group to another, chirality or handedness emerges Alessio Caruana AA with two chiral centres - An amino acid may have more than one chiral centre - This arises when the R functional group in itself has more than one functional group attached to it (e.g. Threonine) Types of amino acids: - The 20 amino acids commonly found in animals are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine - The nature of the side group dictates the role an amino acid plays in a protein - Useful to classify aa according to properties of the side group into: o Polar o Non-polar Types of Amino Acids - Amino acids in biological systems come naturally in L-form - The R groups or side chains determine the physical characteristics o he amino acids and eventually the whole protein - Aliphatic groups are insoluble in water and therefore hydrophobic - They are found deep within the proteins - R group gives rise to Relative Hydrophobicity of an amino acid (e.g. valine (val), leucine (leu), isoleucine (ile) Seven different R groups - These include o Secondary amino groups o Aromatic groups o Aliphatic hydroxy group – medium hydrophobicity (if soluble – low hydrophobicity) o Acidic groups o Basic groups o Amide groups Alessio Caruana o Sulfur groups - The degree of hydrophobicity differs with the different side groups AA with Non-Polar side chains - Each of these amino acids has a non-polar side chain, therefore o They do not bind or give off protons o Do not participate in hydrogen or ionic bonds - In water, these R groups tend to cluster on the interior of the protein - They are usually aliphatic side chains and can associate with oily or lipid-like (lipophilic) therefore hydrophobic interactions - Location of the non-polar side chains determines the eventual function and location of the protein - In aqueous solutions, the side chains of non-polar amino acids o Cluster in the interior of folded proteins o Help to give it the 3D structure o Affect its location in such membranes - In lipid environment of membranes, non-polar side groups o Interact with the lipid o Help localise it as an intrinsic protein - Proline is a special case in that: o The side chain forms a ring structure with the amino group forming an imino group o Its unique structure contributes to the fibrous structure of collagen and interrupts the α-helix of globular proteins - Disulfide bonds are possible through the sulfhydryl group of (-SH) of cysteine. It is an important component of many of the active sites of enzymes. Therefore in cysteine -SH group present - In proteins the SH groups of two cysteines can become oxidised to form a dimer called cystine which contains a disulfide bond or bridge (-S-S-) 5 AA with uncharged polar side chains - These amino acids have a zero net charge at neutral pH though tyrosine can lose a proton at an alkaline pH - Serine, threonine and tyrosine -OH group present - The polar hydroxyl side chain can o Participate in hydrogen bond formation o Serve as points of attachement for other groups - Examples: o Phosphate or oligosaccharide chains in glycoproteins and phospholipids o Serine, threonine and tyrosine kinases - Asparagine and glutamine have a carbonyl and amide group and these also participate in hydrogen bond formation - Asparagine and glutamine contain C=O and NH2 to form the amide group 2 AA with acidic side chains - The side chains of these amino acids donate protons and at neutral pH their side chain is fully ionised and contains a negatively charged carboxylate group (-COO-) - These include aspartic acid and glutamic acid Alessio Caruana - Since they are negatively charged at physiologic pH they are referred to as aspartate and glutamate 3 AA with basic side chains - The side chains of these amino acids accept protons so that at physiologic pH, the side chains of lysine and arginine are fully ionised and positively charged - Histidine is weakly basic and the free amino acid is uncharged at physiologic pH - In proteins its side chain can be either positively charged or neutral depending on the polypeptide’s ionic environment. Acidic and basic properties of AA - Amino acids in aqueous solution contain weakly acidic α-carboxyl group and α-amino groups - They may also contain an ionisable group to its side chain - Thus both free and peptide bound amino acids can act as buffers - The relationship between the concentration of a weak acid (HA) and its conjugate base (A-) is described by the Henderson-Hasselbalch equation - At physiological pH, dissociation occurs - NH3+ and COO- internal ion or Zwitterion is formed - Rate at which Zwitterion forms is dependent on the dissociation constant - If we consider the release of a proton by a weak acid represented by HA or R-COOH: o HA → H+ + A- o Weak acid → Proton + Salt/Conjugate base o R-COOH → - The conjugate base is the ionised form of a weak acid Dissociation constant Ka at Dynamic Equilibrium - At dynamic equilibrium (50% acid dissociation) - Since [RCOO-] = [RCOOH] - Then [RCOO-] / [RCOOH] will cancel out and equal to 1 - Since log 1 is 0, at dynamic equilibrium at which 50% of the acid is dissociated o pK is equal to pH o pH = pKa + 1 (Henderson-Hasselbalch equation) Applications - If a protein is run in a polyacrylamide gel buffered in a salt solution and an electric field applied at opposite ends of the gel, the protein will move either towards the negative or the positive pole - There comes a point where they remain immobile in an electric field and this is called the isoelectric point - All amino acids have their unique isoelectric points Separation of protein molecules by isoelectric focusing [Refer to page 36] Isoelectric focusing - Isoelectric point found: o A tube containing a fixed pH gradient Alessio Caruana o Subjected to a strong electric field in the appropriate direction o Each protein species migrates o Forms sharp band at its isoelectric pH Two dimensional PAGE [Refer to page 36] Some further notes on AA - Amino acids cannot be stored in the body - Excess amino acids are deaminated - Ammonia converted to urea in the ornithine cycle – carbon skeleton - Proteins – only storage polymer for amino acids - Amino acids – used to form hormones, neurotransmitters and some forms of lipids - Body’s nitrogen balance – controlled through a.a. - Only L-amino acids are used by the body - Occurrence of D-amino acids (D-serine in the brain – neurotransmission or neurodegeneration) in the body is not usual (may be due to post translational modifications) pKa and strength of the Acid - The larger the Ka (smaller pKa), the stronger the acid because most of the HA has been converted into H+ and A- - The smaller the Ka (larger pKa) the weaker the acid since less of the acid has dissociated - Therefore amino acids (and other acidic compounds) can be characterised by their pKa value - Remember o Strong aa – small pKa o Weak aa – large pKa Other pK values - pK values are also assigned for other situations - The pK2 refers to the pH at which 50% of the base is dissociated - Since the R groups of some amino acids (polar, acidic or basic side chains) can also dissociate, this R group can be assigned another pK value pK3 - The pK values are unique for each amino acid - Since pK values can be determined by the R group, this can be exploited for protein analysis in electrophoresis. 11th December 2023 BASIC BIOCHEMICALS – PROTEINS Proteins - Group of highly complex organic compounds found in all living cells - The most abundant class of all biological molecules - The most functionally diverse molecules in living systems - Every life process depends on this class of molecules - The specialised study of their mode of action and interactions in living systems has led to a field known as proteomics Alessio Caruana Variety of proteins - Enzymes – key actors in synthesis and metabolism - Polypeptide hormones regulate metabolic processes - Oligopeptides – opioid pentapeptides, antimicrobials - Motor proteins in muscle are responsible for shortening of muscle cells or contractions - Cell support system and cytoskeletal proteins - Scaffolds of collagen fibres with hydroxyapatite salts (calcium and phosphate salts) deposited - Albumin an essential protein in the blood serum - Haemoglobin – RBC protein responsible for the transport of oxygen and carbon dioxide - Immunoglobins and nanobodies – responsible for immunity and to fight infectious organisms Conventional antibodies vs nanobodies - Conventional antibody o Heavy and light chains o Both chains required for antigen binding and stability o Large size and relatively low formatting flexibility o Administered through injection - Heavy-chain antibody o Only heavy chains o Full anigen binding capacity and very stable - Ablynx’s Nanobody o Small (1/10 of a mAb o Flexible formatting o Highly potent, robust and stable o Broad target applicability o Multiple administration routes o Ease of manufacture o Speed of discovery Proteins as macromolecules - Protein comprises approximately 50% of cellular dry weight - Hundreds of protein molecules have been isolated in pure, homogenous form, many have been crystallised - All contain carbon, hydrogen and oxygen and nearly all contain sulfur as well - Some proteins also incorporate phosphorus, iron, zin, and copper at their centre - Proteins are large molecules with high molecular weights (from about 10,000 Da for small ones [50-100 amino acids] to more than 1,000,000 Da for certain forms) Peptide bond formation - A peptide bond is formed when the carboxyl carbon atom of one amino acid covalently binds to the amino nitrogen atom of another amino acid - This is a condensation reaction or dehydration synthesis with the release of a water molecule - Within a protein all (except terminal) carboxyl and amino groups are combined in a peptide linkage - Not available for chemical reaction but still important for hydrogen bond formation - Each amino acid is linked via a peptide link (amide bond) Alessio Caruana - The C-N bond is known as a partial double bond. This gives rise to cis-trans isomerism where the bond cannot move much. The trans isomer form is more stable since cis form gives stearic hindrance Peptide Bond Stability - The peptide bond is considered quite stable an without enzymes estimated to last 1000 years - Prolonged exposure to strong acids and bases at high temperatures hydrolyses bonds - Equilibrium lies on the hydrolysis side rather than synthesis - Therefore synthesis requires energy Proteins as polymers of amino acids - Each amino acid contributes three bonds to the backbone of the chain - By convention, an R group is often used to denote an amino acid side chain - Residues are oriented alternately due to the increase stability of the trans isomer Protein structural components - A proteins consists of a polypeptide backbone with attached side chains - Each protein differs in its amino acids sequence and number - NB Each protein has an amino and carboxyl terminus - It is the sequence of the chemically different side chains that makes each protein distinct - Polypeptides have polarity – the two ends of a polypeptide chain are chemically different o The end carrying the free amino group is the amino terminus or N-terminus o That carrying the free carboxyl group is the carboxyl terminus or C-terminus - The amino acid sequence of a protein is always presented in the N to C direction, reading from left to right Primary structure of proteins - The primary structure of a protein is the sequence of its component amino acids or residues - They can be considered as a linear polymer chain of residues - They may contain hundreds of amino-acid residues arranged in a specific order for a given species of protein - These must eventually be folded to form a three-dimensional structure which is the functional protein. Properties of polypeptides - Regular repeating parts – Main Chain or Backbone - A variable par consisting of distinctive side chains - Stabilised by formation of disulfide bonds and hydrogen bonding in the secondary protein - The main chain – high potential for hydrogen bond formation - Between adjacent residues in adjacent parallel chains Protein secondary structure - Proteins do not assume a random 3D structure - Amino acids located near each other in a linear sequence arrange according to the PHYSICAL INTERACTIONS between a.a. Alessio Caruana - Alterations in the shape of linear sequence is called the secondary structure – helix or zig-zag pattern - Common secondary structure in proteins are o Right handed alpha-helix o Beta-sheet - Less common secondary structures o Beta-bend o Collagen triple helix o Left-handed alpha-helix (less common) - The primary sequence folds in such a way as to form either an alpha-helix or a beta-sheet - Different segments of a primary sequence can be folded into several alpha-helices and several beta-sheets according to its eventual function - For freely soluble proteins the polypeptide chains with polar a.a. usually fold in such a way as to: o Expose polar groups on the outside o Hydro phobic groups are tightly protected on the inside Protein folding for polypeptides - The aa sequence of a protein – folds it into a compact conformation - Polar aa side chains – gather on outside off the protein and can interact with water - Non-polar aa side chains buried on the inside – form tightly packed hydrophobic core hidden from water. a Bonds in secondary protein folding - Three types of non-covalent bonds - Although a single one of these bonds is quite weak, many of them often form together to create a strong bonding arrangement Disulfide bridges and cysteine Alessio Caruana - Cysteine is oxidised to cystine Bovine insulin - An example of intra-molecular and inter-molecular stabilising disulfide bridges evident in functional insulin Polar characteristics of polypeptides - The -NH and -C=O groups of the peptide bond though polar are uncharged - They neither accept nor release protons over the pH range of 2-12 - The only charged group present in a polypeptide are o The N-terminus alpha amino group o The C-terminus alpha-carboxyl group o Any other ionised groups present in the side chains - However, the -NH and -C=O groups are polar - H-bonds occur between the carbonyl oxygen of the peptide bond and the amide hydrogen of another peptide bond. - H-bonds are individually weak but collectively stabilise alpha-helix or beta-pleated sheet - H-bond formation occurs in both in alpha helices and beta sheet structures and these help to give stability to the polypeptide H-bonding - Large umbers of H-bonds between adjacent regions of the folded polypeptide chain stabilise its 3D shape - The enzyme lysozyme – H-bonds between the three possible pairs of partners o Between atoms of 2 peptides o Between peptide bond and aa side chain o Between two amino acid side chains Hydrogen bond potential - In alpha helices the H-bond extends up the spiral from the carbonyl oxygen of one peptide to the -NH of a peptide approximately four residues ahead Alessio Caruana Characteristics of Alpha-Helix - Actually 3.6 aa are accommodated per turn of alpha helix and the pitch is 0.54 nm - System ensures that all peptides except 1st and last are stabilised by H-bonding - Represented as a spiral or cylinder with tightly packed, coiled polypeptide core - Side chains of residues extend outwards from central axis and thus avoids stearic hindrance - Examples of alpha-helix proteins include myoglobin, ferritin, keratin Disruption of alpha-helix - Proline with its imino group inserts a kink in chain and disrupts the smooth helical structure - Charged amino acids such as glutamate, aspartate, histidine, lysine and arginine especially in large numbers also disrupt helix either by: o Ionic bond formation o Electrostatic repulsion - Amino acids with bulky side groups such as tryptophan, valine and isoleucine can interfere with alpha-helix formation especially when found in large numbers H-bonds in the Beta-sheet - Adjacent peptide chains usually run in same (parallel) or opposite (antiparallel) directions - The individual polypeptide chains (strands) in a beta-pleated sheet are held together by hydrogen-bonding between peptide bonds in different chains or segments of same chain - The amino acid side chains in each strand alternately project above and below the plane of the sheet - The H-bonds run perpendicular to the polypeptide backbone, extending between chains Alessio Caruana Beta-bends or hairpins - Beta bends are reverse turns connecting successive strands of antiparallel beta-strands – help form a compact globular shape - Usually found on surface of protein molecules and often include charged residues - Usually composed of 4 amino acids with o One being glycine – smallest R-group o Another being proline – imino group helps to cause the kink - Beta-bends are stabilised by H-bonds and ionic bonds Tertiary protein structure - The tertiary structure of a protein refers to: o The way in which the motifs fold into domains o The final arrangement of domains Super secondary structures - Structural motifs – the way different segments of a polypeptide fold on each other - Form a specific geometric arrangement, e.g. beta-bends are examples of these motifs - They are fundamental structural and functional units of a protein and help create structural domains in a protein - Polypeptides >200 aa usually have two or more domains - Several combinations of super secondary structural motifs possible e.g. beta-alpha-beta or alpha-beta-alpha or beta-5 alphas-beta etc. Motifs vs. Domains - Structural motifs refer to the super secondary structures such as a sequence of alpha helices and beta pleated sheets in a consecutive order Alessio Caruana - A structural domain is a conserved region of a protein’s tertiary structure that can fold independently serves a specific function such as NAD or ATP binding domain present in many enzymes Motifs vs. Domains motif Domain A chain-like biological structure made up of An independent folding unit of the three- connectivity between secondary structural dimensional protein structure elements A supersecondary tructure of a protein A tertiary structure of the protein Formed by the connected alpha-helices and Formed by the formation of disulfide bridges, beta-sheets through loops ionic bonds, and hydrogen bonds between amino acid side chains Mainly have a structural function in the protein Mainly have functional importance structure Have similar functions through protein families Have unique functions Are not stable inndependently Are independently stable Different protein domains Rossman Fold Motif - Protein structural motif of nucleotide-binding protein especially FAD and NAD - Composed of three or more parallel beta strands linked by two alpha-helices – topological order is beta-alpha-beta-alpha-beta Alessio Caruana - Each Rossman fold can bind one nucleotide – single Rossmann folds can bind mononucleotides such as the cofactor FMN - For dinucleotides such as NAD binding domains consists of two paired Rossmann folds that each binds one nucleotide moiety 18th December 2023 Quaternary Protein structure - The quaternary structure refers to the way several polypeptides interact together to form the functional protein e.g. Immunoglobins Functional Quaternary structure - Haemoglobin consists of two alpha and two beta globin chains each with a haem prosthetic group Conformational changes to a Protein Quaternary structure Types of Proteins - There are many different types of proteins - The main characteristics are dictated by: o The function of the protein – structural, enzymatic, etc. o The interactions with other molecules o The final environment of the protein – whether extracellular or intracellular. Intracellular proteins - The intracellular environment is a mildly reducing environment - Includes cytosolic and nuclear - Many are globular and soluble - Often oligomeric - Mostly all-alpha or alpha-beta (alternating) - Most cysteines have free sulfhydryl groups -SH (or thiol C-S-H group) Extracellular proteins - The extracellular environment is oxidising - Also included are interior of lysosomes and endoplasmic reticulum - P mostly all-beta or mixed alpha and beta - Exception – 4-helix bundles in cytokines - Many are globular and soluble - Most cysteine amino acids are oxidised into cystine form with disulfide bridges Types of proteins - Different classes are recognised some of which form the bulk of proteins present in living organisms o Integral membrane proteins o Structural proteins ▪ Intracellular ▪ Extracellular o Viral proteins and transcriptases Alessio Caruana o Enzymes o Ancillary types – carriers, hormones o Periplasmic proteins o Foldases and chaperones Integral membrane proteins - Photosynthetic reaction centre bacteriorhodopsin - 7-trans membrane helix - G-protein-coupled receptors - Nicotinic acetylcholine receptor - Proton and ion pumps - Voltage-gated ion channels - Gap junction proteins, etc. Structural proteins - Intracellular o Muscle proteins, actin, myosin, dystrophin, troponins o Cellular cytoskeleton, F- and G-actin, dynein, microtubules, ankyrin, vilin, etc. - Extracellular o Extracellular matrix, collagen, fibronectin, laminin, vitronectin, fibrinogen, fibrin, titin, twitchin o Adhesion proteins, glycoproteins, keratin, silks Collagen: structural protein - Collagen is a triple helix formed by three extended protein chains tha wrap around one another - Many rodlike collagen molecules are cross-linked together in the extracellular space to form inextensible collagen fibrils - The striping on the collagen fibril is caused by the regular repeating arrangement of the collagen molecules within the fibril Elastin Structural protein - Elastin polypeptide chains are cross-linked together to form rubberlike, elastic fibres - Each elastin molecule uncoils into a more extended conformation when the fibre is stretched - It recoils spontaneously as soon as the stretching force is relaxed Viral proteins - Coat proteins – a helical coat constructed from repeated copies of a coat protein 158 amino acids long (capsomeres) Ligand binding proteins - Many weak bonds are needed to enable a receptor protein to bind tightly to a second molecule, which is called a ligand for the protein - A ligand must therefore fit precisely into a protein’s binding site, like a hand into a glove - A large number of non-covalent bonds can be formed between the protein and the ligand Alessio Caruana The binding site of a protein - The folding of a polypeptide chain typically creates a crevice of cavity on the protein surface - This crevice contains a set of amino acid side chains arranged in such a way that they can made non-covalent bonds only with certain ligands. - A close-up of an actual binding site showing the hydrogen bonds and ionic interactions formed between a protein and its ligand - E.g. cyclic AMP is the bound ligand Enzymes - Thousands of enzymes that catalyse all the biochemical pathways - Mostly in cytosol – transferases, hydrolases, lyases, isomerases, etc. - Many are variations on basic Rossman-fold (as in lactate dehydrogenase) - Many use ATP and/or other cofactors, such as metal ions, NAD, FAD, etc. - Often multimers with allosteric cooperative binding – change shape between active and inactive state. Kinases and Phosphatases - Proteins are modified by the covalent addition of a phosphate P gr. - Transfer of a P gr. From ATP to an aa side chain of the target protein catalysed by a protein kinase o The phosphate is added to a serine side chain o The phosphate can be linked to the -OH group of a threonine, serine, or a tyrosine in the protein - Removal of P gr. Catalysed by a protein phosphatase - The phosphorylation of a protein by a protein kinase can either increase or decrease the protein’s activity, depending on the site of phosphorylation and the structure of the protein Cell protein pathways - The MAPK has role of transmitting signals from cell surface receptor to DNA - Uses a series of different proteins some of which are phosphorylated in the process: o Growth factor receptors o Small G-protein RAS o Docking protein GRB2/SOS o Signal transduction proteins o Final transcription protein CREB/MYC Receptor Tyrosine Kinase Cascades - A particular receptor may affect more than one cascade - Certain parts of different cascades may be interlinked - Note arrow – cycle continues, stop notation – cycle blocked/protein inhibited Types of enzymes - Nucleic acid manipulation and regulation proteins o Polymerases, nucleases, ligases, gyrases, topoisomerases o DNA-binding proteins, transcription factors, zinc-fingers, repressors, histones, steroid receptors - Response Elements – some of these are membrane associated Alessio Caruana o Elements of 2nd messenger pathways – kinases, phosphatases o Small G-proteins, adaptors (SH2, SH3, PH domains), etc. o Calcium ions and phosphate groups play a large role here. Calcium ions activate lipases and calmodulin - Redox and electron transport proteins o Dismutases, cytochromes, thioredoxin, ferredoxin, etc. o Haem groups and metal ions often employed as cofactors - Hydrolase enzymes – breakdown of bonds to form smaller subunits o May be both intercellular and extracellular enzymes o Several different groups that are named after their different substrate o Differ on site of action – internal or external in macromolecule Hydrolase enzymes – 4 basic types - Nucleases cut nucleic acids (e.g. pancreatic RNase) - Lipases cut and process lipid chains - Glycosidases cut sugar moieties (e.g. lysozyme) - Peptidases (proteases) – help to cleave one or a limited number of peptide bonds Peptidases (Proteases) - Subdivided into two groups o Exopeptidases – cleave bonds and remove amino acids sequentially from either the N- or C-terminus o Endopeptidases (Proteinases) – cleave bonds at points within the protein Endopeptidases (Proteinases) - There are 4 main classes of endopeptidases o Serine proteases ▪ Prokaryotic serine proteases e.g. subtilsin ▪ Mammalian serine proteases e.g. trypsin, chymotrypsin and elastase o Aspartic proteases e.g. chymosin, pepsin, renin o Cysteine proteases e.g. papain, actinidin o CASPASES – cysteine proteases that cleave after an aspartic acid residue o Zinc metalloproteases e.g. thermolysin, carboxy-peptidase A Ancillary types - Binding proteins and adhesion domains – e.g. fibronectin type I and II - Storage and carrier proteins, e.g. albumin, retinol-binding protein, ferritin - Hormones (e.g. insulin), cytokines and growth factors (TGF, EGF) - Receptor domains - Recognition/Immune response proteins, e.g. immunoglobins, lectins - Complement proteins (MHC – Major Histocompatibility Complex) Periplasmic proteins - The space between the bacterial membrane and its outer cell wall is somewhat special - Same applies for mitochondrial matrix and intermembrane space - A number of specialised binding and transport proteins are found in these spaces - These include: Alessio Caruana o Arabinose-binding protein o Sulfate-binding protein o Phosphate-binding o Leu-Ile-Val-binding protein Foldases and Chaperones - Proteins that assist correct folding of polypeptides Types: o HSPs – called heat shock proteins – upregulated during heat shock - Several classes – HSP 70, 40, 90 systems and HSP60/10 o HSP70: protects an unfolded protein from degradation folding, HSP40 and HSP90 act as cofactors small HSP o HSP60/10 or Chaperonins prevent mis-aggregation, actively help protein folding o Peptidyl-prolyl isomerase assists in converting cis-proline to trans o Disulfide isomerase helps to shuffle cysteines paired in disulfide bridges until the ‘correct’ linking is achieved Proteasomes - Proteasomes are large protein complexes - Found inside all eukaryotes, archaea and bacteria - In eukaryotes, they are located in the nucleus and cytoplasm - Function of proteasome – to degrade damaged proteins (Ubiquitin tagged) by proteolysis - 26S proteasome: 20S core and 19S regulatory particles at the end - Needs ATP to function Proteins in apoptosis - Many different types of proteins involved - These include: o Receptors – death domain o Caspases – initiator and effector o Scramblases or Flippases o Apoptosome – Apaf 1 and Apaf 2 o IAP – inhibitors of apoptosis o Endonuclease G o AIF apoptosis inducing factor Caspases - A family of cysteine aspartase proteases - Cleave proteins after an aspartic acid residue - Hence cysteine-aspartic-acid-proteases - Have conserved aa sequence (QACRG – glutamine, alanine, cysteine, Argentine and glycine) - They are potent intracellular proteases that will dismantle the cell - Currently 11 human caspases have been described namely caspase 1-10 and 14 - Caspases act at two levels – the initiator and effector (executioner caspases) Intrinsic and extrinsic apoptotic pathways - Apoptosis mediated through extrinsic and intrinsic pathways – involves initiator and effector caspases Alessio Caruana - Extrinsic pathway involves a death receptor to recruit and activate the initiator caspase 8. - Intrinsic pathways involve release of initiator procaspase 9 and cytochrome C, endonuclease G and AIF from the mitochondria - Procaspase 12 in mice (or procaspase 5 in humans) is released from the RER - Activated initiator caspases act on effector caspases 3, 6, and 7 to execute apoptosis. Apoptosome - The release and activity of caspases is tightly regulated by inhibitor of apoptosis (IAP) - A potent catalyst of apoptosis is cytochrome C (Apaf-2) which pairs up with apoptotic protease activating factor one (Apaf-1), a cytosolic scaffolding protein - These associate into a heptameric, ATP-stabilised apoptosome Stearic limitations on bond angles - The peptide bond is planar – being a partial double bond is rigid and does not permit rotation - By contrast, rotation can occur about the Cα-C bond, whose angle of rotation is called psi, and about the N-Cα bond, whose angle of rotation is called phi - The conformation of the main-chain atoms in a protein is determined by one pair of phi and psi angles for each amino acid as illustrated in the Ramachandran plot - Each dot represents and observed rotation of the protein. 8th January 2024 CELL MEMBRANES – STRUCTURE AND FUNCTION Lipids & Proteins interactions - Some membrane proteins have been shown to adhere to the hydrophobic fatty acyl cores of the bilayer - Others bind to the membrane by protein-protein interactions - Specialised proteins fulfil specific membrane functions but structural integrity is mainly due to properties of lipids - Several factors affect fluidity of a bilayer: o Lipid composition o Cholesterol content o Temperature Lipids & Membrane Physical Properties - As a lipid bilayer made from a single type of phospholipid is heated, there is a point when: o An abrupt change in the physical properties of the lipid bilayer occurs o Characterised by an ordered gel (crystal) state transition to a disordered fluid (liquid crystal) state o This happens over a narrow temperature range called phase transition o It occurs due to the reorganisation of the fatty acyl side chains Phase transition - Phase transition is the change from a highly ordered, gel (crystal) state to a more mobile fluid state (Liquid crystal) Alessio Caruana - Increased motion about the C-C bonds of the fatty acyl chains are responsible for change - Allows them to assume a more random orientation - Short chain fatty acyl chains have less surface area with which to form van der Waals interactions with each other – tend to maintain a more fluid state - Unsaturated fatty acyl chains have kinks that similarly cause less stable van der Waals contacts and thus tend to maintain a more fluid state - Gel-fluid transition is accompanied by absorption of a high amount of thermal energy - Lipids with short or unsaturated fatty acyl chains (Fewer vdW) undergo fluid to gel phase transition at very low temperatures. Membrane does not freeze at below zero temperatures - Lipids with long or saturated fatty acyl chains (more vdW) undergo fluid to gel phase transition at relatively high temperatures. Membrane can freeze at below zero temperatures - How can we relate this to organisms living in very cold environments? Membrane fluidity - For biological cell membranes, fluidity is essential to normal growth and reproduction - The mixture of fatty acyl chains in biological membranes ensure membrane is fluid at the temperature at which the cell is growing. - As environmental temperature decreases, a proportional increase in unsaturated fatty acyl chains occurs, thus maintain the fluidity at lower temperatures - Hence, organisms introduce more PUFA (22:6) as temperature falls or in highly active cells Membrane fluidity and cholesterol - Cholesterol – major determinant of membrane fluidity - Cholesterol is too hydrophobic to form a sheet structure on its own – can intercalate with other phospholipids to alter its physical properties - The polar hydroxyl group of cholesterol interacts with the polar head group of the phospholipid - BUT steroid ring tends to immobilise part of the fatty acyl chains Cholesterol in a lipid bilayer [insert diagram from Mol Bio of the Cell] Membrane fluidity and cholesterol - Cholesterol restricts art of the fatty acyl chain closest to the leaflet surface - Separates and disperses fatty acyl chains and causes inner regions of bilayers to become slightly more fluid - At temperatures below phase transition cholesterol keeps the membrane fluid by preventing the hydrocarbon fatty acyl chains from binding each other - BUT excess cholesterol makes eukaryotic CM less fluid at 37°C, and introduces problems within cells – e.g. enhances ion permeability Cell membrane common features - Sheet-like structures typically 6-10 nm thick - Composed of protein and lipids held together by non-covalent interactions - Highly selective permeability barriers - Create closed compartments – entire cell or for subcellular structures - Proteins in membrane are responsible for regulation of ion composition of compartments Alessio Caruana - Proteins also help in regulatory and catalytic action - Membranes also control flow of substances and chemical messages between cells Membranes are highly dynamic FRAP - Limited natural diffusion between bilayers – 1 every couple of hours - Continuous state of motion in individual layer – monitored by electrospin resonance (ESP) and fluorescence recovery after photobleaching (FRAP) Basic concepts 1 - Addition of new phospholipids – on the cytosolic monolayer side of the endoplasmic reticulum membrane i.e. in only one layer o Phospholipid translocators or Flippases needed to transfer selected phospholipids to non-cytosolic side o Scramblase transfers PL in both directions - Substantial influence of cis-double bond in fatty acyl chai on membrane fluidity o Introduce kinks in chain and make it more difficult to pack chains together o PL are more spread out, bilayer is thinner Basic concepts 2 - PUFA is a compensation for decreased environmental temperatures BUT: o High PUFA content enhances cell membrane permeability properties especially to Na+ ions o The Na+/k+ pump has to be more active especially in membranes of neurons and other nervous tissue o Higher PUFA increases chance of free radical damage and membrane degradation – membrane peroxidation o Cholesterol ALSO enhances ion permeability properties like PUFA o In eukaryotes 1:1 cholesterol to PL ratio is common Lipid bilayer asymmetry - Difference in lipid composition of the monolayers - Lipid head groups asymmetry in human RBC o In outer leaflet have phosphatidylcholine (PC) and sphingomyelin (SM) o Inner leaflet phosphatidylethanolamine (PE) and phosphatidylserine (PS) - PS net negative charge – hence also charge asymmetry - More saturated fatty acids in outer monolayer than in inner monolayer – could there be an explanation? - Has functional importance – cytosolic proteins bind specific lipid head groups e.g. protein kinase C (PKC) which requires a negative charge for its activity (PS) - Phospholipases are present that are activated by extracellular signals (e.g. phospholipase C acts on inositol to activate PKC and to stimulate release of Ca2+ ions - PS translocation also important during apoptosis Glycolipid asymmetry - Sugar-containing lipids (around 5%) on non-cytosolic side - GL tend to self-associate preferentially into rafts - Sugars added in lumen of Golgi apparatus Alessio Caruana - GL neutral -Galactocerebrosides or (-) charged gangliosides - In bacteria, GL derived from glycerol whilst in animal cells always produced from serine - Protect cell from harsh conditions or alter membrane electrical properties – GL on apical surface of epithelial cells of intestine - Cell-to-cell recognition and adhesion – carbohydrate binding proteins (lectins) bind GL and glycoproteins - Certain bacteria use host GL as entry points o Part of cholera bacterial toxin binds the GM1 ganglioside o Other part enters the cells and binds and stabilises small G proteins o Prolonged intracellular cyclic AMP activity o Large efflux of Na+ and water into intestine o Leads to dehydration and possibly death Membrane proteins - Protein and lipids have a 1:1 ratio vy mass but there are around 50 lipid molecules to one protein molecule - Proportion and types of proteins highly variable: o Myelin (mainly insulating)

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