Intro to Protein Biochemistry PDF

*[Molecular basis of inheritance ]* *[Intro to protein biochemistry]* Proteins are very diverse molecules and have a wide variety of structures and so a wide variety of functions. - Hormones: small proteins that travel around the bloodstream and bind to specific receptors elsewhere in the b...

*[Molecular basis of inheritance ]* *[Intro to protein biochemistry]* Proteins are very diverse molecules and have a wide variety of structures and so a wide variety of functions. - Hormones: small proteins that travel around the bloodstream and bind to specific receptors elsewhere in the body - Antibodies: recognition of foreign material, allowing the immune system to respond - DNA binding proteins: bind to specific DNA sequences and affect gene expression *[Movement of other molecules (large structures)]* - Porin- sits in outer membrane of bacteria allowing diffusion of certain molecules - Ferritin- stores, transports and releases iron (single protein copy binded to many others) *[Structural functions]* Proteins form various components in the cytoskeleton, e.g. microtubules. These are formed from many alpha and beta tubulin subunits, used to maintain overall structure of the cell. Very strong yet dynamic and have a large structure. They are also involved in mitosis, separating chromosomes for division, and occur and the base of cilia and flagella as helper cells *[Enzymes]* These are proteins that accelerate the rate of chemical reactions and do not change final equilibrium. This is done by reducing energy needed to carry out reaction. Proteins have an active site that binds to the substrate and yields a product. *[4 levels of protein structure]* Proteins are made of amino acids with different side chains that are joined to make polypeptide chains. Side chains can have different shapes, sizes, electric charges and polarity. They have 1 letter or 3 letter codes *[Primary structure]* - This is the order of amino acids; protein sequence is defined by the gene sequence- 3 DNA bases =1 amino acid - Amino acids are joined by peptide bonds, losing a water molecule (condensation reaction) - Generally, 50-2000 amino acids in a protein structure but can be much longer. Shorter chains are called peptides rather than proteins. - Rotation is not possible around peptide bonds *[Secondary structure]* - Proteins folded into a 3d structure- moving from denatured to native protein (folded) - Denatured proteins are simple chains and very thermodynamically unstable- bonds form between amino acids, causing them to fold - Some proteins fold spontaneously, and some require "chaperones" to help them fold into the right shape Hydrogen bonding is a weak interaction but is strong when many interactions occur at once - Alpha helix- hydrogen bonds form between amino (+) and carboxyl (-) groups of amino acids, groups if 4 residues apart, forming a helical shape - Beta sheet- hydrogen bonds between amino and carboxyl groups that are further away, on different strands, causing a pleated shape- can be parallel or anti parallel Structures can be drawn using ribbon diagrams *[Tertiary structure]* - tightly packed, thermodynamically stable 3d structure of the protein - determined by non-covalent interactions between the side chains, can be interactions of different strength, all non-permanent bonds e.g. hydrophobic (no polarity) vs hydrophilic - electric charges cause side chains to either repel or attract - size and shape of side chains will attribute to shape and folding Different amino acids interact differently with polar water molecules, so side chains can form hydrogen bonds. Hydrophobic amino acids tend to be on the inside of the tertiary structure, away from the aqueous environment of the cell. Disulphide bridges can form between cysteine residues - interactions between sulphur atoms on the side chain cysteine - causes crosslink to form between different parts of the primary sequence via oxidation Some proteins can fold into a single compact structure but many fold into several domains (tightly folded regions), separated by flexible regions that are less tightly folded Domains often carry out a specific part of the proteins function, however the same domain can appear in several evolutionary linked proteins *[Quaternary structure]* Sometimes polypeptides come together to form a more complex structure with two or more subunits. This is the quaternary structure e.g. haemoglobin - dimer= two - trimer=three - tetramer=four *[Post translation modifications]* - Removal of specific parts of the sequence e.g. signal peptides - Addition of molecules 1\. Methylation- reversible addition of -ch3 groups, methylation of histones control gene expression 2\. glycosylation-addition of various sugars, especially on the cell surface and secreted proteins (reversible) 3.ubiquitination- addition of 76 amino acid polypeptide called ubiquitin. This marks a polypeptide for degradation. This is not reversible *[Phosphorylation]* - Reversible addition of a phosphate (po3) by a class of enzymes called kinases - Important way of regulating enzyme function - Phosphorylation of amino acids in or around active site can change properties of region and alter substrate binding *[Protein targeting]* - different organelles require different types of proteins - some proteins need to be moved to the membrane or secreted from the cell - many proteins contain a short signal or localisation sequence showing where they need to go - some proteins are completely synthesised in the cytoplasm and then delivered to their desired location Some proteins can be targeted to cell membrane for secretion. This occurs via the secretory pathway; ribosomes associate with the endoplasmic reticulum. Some proteins can span the membrane- hydrophobic regions go inside the membrane, hydrophilic parts go on inside and outside and interact with soluble regions Others associate with a trans membrane protein or directly with the polar heads of the membrane lipids *[Anchoring membrane proteins]* - Other proteins are anchored in the membrane by addition - hydrophobic groups added to protein sequence - E.g. addition of fatty acid groups to small g proteins - When at the membrane- involved in signalling pathways - Modification allows them to be removed from the membrane- inactive in the cytosol - Cycle on and off the membrane *[DNA as the genetic material]* Mendelian laws of inheritance: - Segregation: genes come in pairs, and individuals only pass on one of these to their offspring - Independent assortment: different genes are passed on separately from each other, inheritance of one does not depend on the other - Dominance: an individual with two alleles of a gene will express the dominant form So, there must be some physical form of genetic material "Information that, when passed to a new generation, influences the form and characteristics of each individual" - Replication of information - Storage of information - Expression of information - Variation by mutation *[Sutton-Boveri theory of chromosomal inheritance]* - Used two advances in microscopy to observe early development in 2 organisms and their chromosomes. - Chose their model organism due to small number of large chromosomes, so are easy to observe. - Used cytology and microscopy to test this "Where is the genetic material carried?" Chromosome theory of inheritance provided a physical basis for Mendel's independent assortment - Chromosomes are required for embryonic development - Chromosomes carry Mendel's "factors" (genes) - Chromosomes are linear structures with genes along them *[The transforming principle]* Frederick Griffith- streptococcus pneumoniae can cause pneumonia in humans and mice. Only some strains cause infection and illness, some living in the nasal passages without causing disease. - **S strain** -- smooth bacteria- pathogenic- polysaccharide capsule- protects from host immune system - **R strain**- rough bacteria- not pathogenic Test to transform rough bacteria into smooth 1. Kill **S** bacteria by heating them to high temperatures, no infection established, mouse is not infected 2. Inject dead **S** bacteria into mice together with live **R** bacteria, mouse is now infected 3. Interaction between **S** extract and **R** strain, live **S** strain is recovered from mouse with capsule *[Results]* Inoculation with dead **S** bacteria and live **R** bacteria establishes infection. Bacteria taken from infected mice do have a polysaccharide capsule, therefore **R** cells have undergone a "transformation". Some sort of hereditary material has passed from the **S** bacteria to **R** bacteria, changing the genotype. The is called the transforming principle. - Avery, MacLeod, McCarthy Systematically destroyed each component of the s strain extract using enzymes that specifically digest each type of molecule, before combining with R to find out which is responsible. *[Candidates for the transforming principle:]* - Protein -- complex and abundant - DNA- too simple - Polysaccharide - Lipids - RNA For the bacteria to be virulent, they require the DNA encoding for the enzymes to make to polysaccharide coat but not the polysaccharide themselves. DNA from **S** cell extract is being taken up by different individual **R** cells. The few cells that take up the gene coding for the polysaccharide capsule will become virulent. Other bacteria with different integrated genes will not be virulent *[Bacteriophage genetic material]* Hershey and Chase Which component of a bacteriophage is injected into bacteria? What they knew: - The basics of bacteriophage life cycle - The basics of the chemical composition of DNA and proteins What is a bacteriophage? - A category of viruses - All viruses require a host cell to reproduce - Host cell for bacteriophage is a bacterium For T2, the bacteriophage host is specifically the bacterium E. coli T2 life cycle: - DNA genome, destruction of the hosts cell's chromosomes - Transcription and translation of viral genes and replication of the viral genome - Assembly of new virus particles from DNA and protein subunits *[Outline of the experiment:]* Phage ghost- phage stuck to outside of bacterium after injecting DNA into it 1. Label bacteriophage DNA or protein with a radioactive isotope - ^32^P and ^35^S are unstable isotopes of phosphorous and sulphur. They knew that DNA contains phosphate groups and proteins contain sulphur. These isotopes can be detected by Geiger counter. - Growing bacteriophage in media containing ^32^P will yield phage containing radioactively labelled DNA - Growing bacteriophage in media containing ^35^S will yield phage containing radioactively labelled protein 2. Infect unlabelled bacteria with radioactive phage - They knew that only the genetic material entered the infected cell- the rest of the virus remained attached to the outside of the bacterial host, allowing them to follow where the DNA or protein went 3. Separate the phage ghosts from infected bacteria - Disrupt in blender to make ghosts fall off bacteria. Bacteria remain intact. 4. Test bacteria and phage ghosts for radioactivity - Separate by their weight (bacteria are heavier) using centrifuge - Pellet contains bacteria (including the phage genetic material) - Supernatant contains phage ghosts. - Test both fractions for radioactivity with a Geiger counter. 5. Are bacteria or the phage labelled? - If DNA or protein were in infectious material, they would be in the bacteria results show that DNA is carried over from generation to generation. *[Experimental data on DNA structure]* - DNA is made up of nucleotides (phosphate + deoxyribose + base) - These nucleotides are joined by covalent bonds - Nucleotides form chains by phosphodiester linkage *[Erwin Chargaff]* - Inspired by Averys paper showing that DNA transforms bacteria. - Used paper chromatography to separate and isolate the nucleobase components of DNA from a number of species. - Shows that there's equal numbers of purines and pyrimidines. Chargaff's rules: - A%=T% and G%=C% - OR %purines=%pyrimidines - \%AT=/=%GC Composition varies between species; some species are more "AT" rich or "gc" rich *[X-ray crystallography structure of DNA]* - Create a pure solution of molecule of interest - Create a crystal lattice - Shoot x-ray through - Capture pattern that occurs on photographic plate- should by even pattern since crystal absorbs and refracts ***Maurice Wilkins and Rosalind Franklin*** - x pattern showed that the structure was a helix - Regular pattern showed that there was a repeating + even structure - Distance between spots=distance of one turn (3.5nm) - Spots come from DNA molecules refracting x-ray ***Watson and crick*** Information they had: - Structure of nucleotides - Ratios of different nucleotides in the DNA molecule - Crystal structure Main features - A-T and G-C hydrogen bonded pairs H-bonding occurs between a purine and a pyrimidine due to an attraction between slightly negative charged atom (often oxygen) and slightly positive charged atom (often hydrogen). One purine and one pyrimidine maintain the even width of the DNA molecule Mispairing two purines or two pyrimidines would cause distortion in the DNA molecule. - Antiparallel strands Strands all have a 3'-5' polarity which occurs in opposite directions on both strands of DNA. Antiparallel complementary DNA strands will twist around each other to create a double helix - Right-handed double helix This is due to the 2 strands having a phosphate backbone that creates a spiral shape, so double helix occurs - One helical turn every 10.5bp - Major and minor grooves Since there is one turn every 10.5 bp, twisting isn't even. This creates grooves in the sides of DNA *[Structural elements of chromosomes and plasmids. ]* Chromosome- a long DNA molecule with part or all of the genetic material of an organism. Most eukaryotic chromosomes include packaging proteins called histones. Sutton and Boveri showed that chromosomes contained genes along their length, along with regulatory sequences and other important structural elements [Eukaryotic chromosomes ] - Linear molecules, held in the nucleus - Often many chromosomes per genome - E.g. human genome- typically 22 pairs of chromosomes= 2 sex chromosomes between 50 mil and 2.5 bil base pairs Centromeres - Doesn't have to be in centre of chromosome - Variety of DNA sequences throughout eukaryotes but conserved histones - Centromere= the specialised chromosomal region upon which the structures that link to spindle microtubules assemble and direct the equal segregation of chromosomes during mitosis and meiosis Telomeres - protect the wends of linear chromosomes - the repetitive DNA and the ends of linear chromosomes Prokaryotic genomes - Bacteria generally have a single circular chromosome - Typically around a few billion bp large - Plasmids are also found- circular molecules but only a few thousand bp - Plasmids can carry a variety of advantageous genes such as antibiotic-resistant cassettes - They are passed between cells via conjugation. DNA binding proteins - DNA binding domains in proteins - General affinity for DNA or sequence specific - Can prefer single or double stranded DNA - Roles of DNA binding proteins - Regulate gene expression- inform which genes are transcribed - Cut DNA at specific sequences - Protects DNA in various ways Example- transcriptional regulators - Proteins that bind regulatory sequences near to the promoters of genes to either stimulate or block transcription. Bend the DNA into a favourable or unfavourable shape - Lac operon in E.coli- lac repressor binds to DNA to block transcription(when lactose is absent), catabolic activator protein binds to DNA and increases transcription (when glucose is absent) Example- restriction endonucleases - Enzymes that cut DNA at specific, normally palindromic sequences of 6-10 bp - Originated in bacteria to restrict the action of viruses - Viral DNA is cut by the enzyme, but bacterial DNA is methylated and therefore protected - Used by scientists to manipulate DNA Example- histones - Eukaryotic genomes are packaged in chromatin - DNA is wrapped around proteins called histones - Generally non sequence specific *[DNA replication]* -semi conservative \- if we know what's on one strand of DNA, we should know what's on the other- due to base pairing *[Possible models for DNA replication ]* Meselson and Stahl - Used difference between isotopes to do their experiment - They used nitrogen since DNA contains lot of it - Extra neuron means that ^15^N is heavier than ^14^N. - DNA molecules containing the different isotopes can therefore be separated by their weight - These are NOT radioactive *[The experiment]* - Grow bacteria in media containing ^15^N: make "heavy" DNA - Transfer them to media containing ^14^N: new DNA will be "light" - Separate heavy and light molecules by ultracentrifugation Using UV light we can see the weight of the material after ultracentrifugation. Less dense material will reside at the top of the tube and more dense material will be at the bottom. Ultracentrifugation- works with a dense liquid. Sample mixed with liquid, centrifuged very fast for long time, which separates low and high density. *[results ]* - a single hybrid band in the tube discounts the conservative model - 2^nd^ gen- bands in med and top of tube- discounting dispersive model  *[The process of replication]* Single origin of replication in E. coli genome vs tens of thousands of origins of replication in the human genome "Bidirectional replication forks " *[Enzymatic activities for DNA replication ]* - Primase - DNA polymerase - DNA ligase - Topoisomerase - Helicase - Single strand binding protein DNA polymerase - Add nucleotides one at a time, in a 5'-3' direction - Using template strand to form H-bonds tells which base to add next - Tens or hundreds of nucleotides per second - Primase generates the primer, and ligase joins the stretches of new DNA together. Primase makes the primer out of RNA (usually), and DNA poly7merase can add on to the 3' end of the primer. - Ligase joins loose ends together into a single strand of DNA - Base pairing between nucleobases but gaps in the sugar phosphate backbone must be joined up (by enzyme). - Topoisomerase relieves pressure from overwinding around the replication bubble by making and resealing breaking in DNA - Helicase breaks hydrogen bonds between the two DNA strands, separating them. - Single strand binding protein binds to separated strands, preventing them from re-annealing *[Leading and lagging strands of DNA replication]* - Leading strand: 5'-3' DNA synthesis points towards the replication fork and can proceed continuously - Lagging strand: 5'-3' DNA synthesis points away from the replication fork and must therefore be discontinuous (primed numerous times) - Okazaki fragments = the pieces of DNA that are stuck together to make up the lagging strand of replication - DNA polymerase ALWAYS adds on to the 3' end!! - Semi-discontinuous DNA replication - built in several pieces - ligase fills gaps *[Erosion of genetic material at ends of linear chromosomes ]* - Primer removal at the end of the chromosome leaves a gap that can't be filled in (there is no DNA polymerase coming along to fill in that piece). - So on every round of replication, a little piece is lost from the end of the chromosome. - This is a problem for the lagging strand at each end of the linear DNA. - This problem is solved by telomeres - Telomeres = short DNA sequences that are repeated over and over at the ends of the chromosomes. - Short stretches are lost from telomeres at each round of replication. - But that's alright because there is an enzyme called TELOMERASE that can replenish the telomeres from an RNA template in some cell types - The repetitive nature of telomeres also allows them to bind the shelterin complex of proteins, protecting the vulnerable ends of the chromosome and differentiating them from DNA breaks.