Prelim Biochemistry PDF

TWO GENERAL TYPES OF CELLS - includes bacteria, cyanobacteria 1. EUKARYOTES - have a membrane-enclosed nucleus encapsulating the DNA - ncludes higher organisms: plants...

TWO GENERAL TYPES OF CELLS - includes bacteria, cyanobacteria 1. EUKARYOTES - have a membrane-enclosed nucleus encapsulating the DNA - ncludes higher organisms: plants, animals, fungi, protozoa, most algae 2. PROKARYOTES - lack a discrete nucleus and no nuclear membrane MAJOR ORGANELLES OF EUKARYOTIC CELLS - ROUGH ENDOPLASMIC 1. PLASMA MEMBRANE RETICULUM: Lined on the - Holds the cell together cytoplasmic surface with - Serves as selective barrier ribosomes. Site of protein permitting entrance of synthesis. For storage and essential nutrients and secretion of protein products preventing loss of needed outside the cell substances - Secretes waste products - SMOOTH ENDOPLASMIC - Binds certain regulatory RETICULUM: Absence of substances through its ribosomes. Enzymes present receptors differs according to function of the system 2. NUCLEUS - Repository of genetic 1. In intestinal cells - for synthesis of information; information is triglycerides encoded in the base sequence of DNA molecules 2. In adrenal cortex cells – for of the chromosomes synthesis of steroid hormones - NUCLEOLUS: Site of 3. In liver – site of cytochrome P450 ribosomal assembly. which is involved in the metabolism Contains copies of genes for of steroids, drugs, etc. rRNA 4. GOLGI APPARATUS 3. ENDOPLASMIC RETICULUM - Flattened sacs or vesicles - Array of vesicular spaces continuous with ER to which separated from cytoplasmic the newly synthesized fluids or cytosol by a system proteins are transported and of membranes temporarily stored - Site of glycosylation  spindle. Constituent of cilia - MICROFILAMENTS: Consist 5. MITOCHONDRIA of actin. Form contractile - Site of cellular respiration assemblies that is and production of ATP responsible for intracellular - Number and size may reflect movements the need for energy and particular nature of - INTERMEDIATE metabolic activity occurring in FILAMENTS: Prominent in the tissue parts of the cell subject to - Contains the enzymes of mechanical stress citric acid cycle, - B-oxidation of fatty acids, 9. CYTOSOL electron transport chain and - Aqueous matrix that remains oxidative phosphorylation after the insoluble - Contain their own DNA and components of the reproduce by dividing in two cytoplasm are removed - Metabolic processes that 6. LYSOSOMES occurs in the cytosol: - Vesicles surrounded by - Glycolysis, gluconeogenesis, membrane containing pentose phosphate pathway, hydrolytic and degradative activation of amino acids, enzymes biosynthesis of fatty acids - Functions to digest material ingested by endocytosis and recycle cellular components PLANT CELLS 1. CELL WALL 7. PEROXISOMES - Major component is cellulose - Also microbodies - Accounts for structural - Contain oxidative enzymes strength of plants (oxidases) and catalase which synthesize and 2. CHLOROPLAST degrades hydrogen peroxide - Resembles mitochondria - Function to protect sensitive - Stroma encloses cell components from interconnencted stacks of oxidative attacks sacs called thylakoids which contain chlorophyll 8. CYTOSKELETON - Generate ATP to drive - Array of filaments; give the photosynthetic reaction cell its shape and ability to forming carbohydrate and move; responsible for the other products arrangement and internal motion of its organelles 3. VACUOLE - Typically occupy 90% of - MICROTUBULES: volume of mature cells Composed of the protein - Storage depots for nutrients, tubulin. Form the supportive waste and specialized framework that guided the materials such as pigments movement of organelles within a cell, eg, mitotic SUBCELLULAR FRACTIONATION Process of isolating specific ORGANELLES MARKER organelles in relatively pure form, free of contamination by other organelles Nucleus DNA STEPS Mitochondria Glutamic 1. EXTRACTION Dehydrogenase - Avoids extreme pH and osmotic pressure and high Endoplasmic Glucose temperature Reticulum 6-phosphatase - Employs aqueous solution - T = 0 – 4oC; to avoid loss of Lysosomes Acid Phosphatase biologic activity Plasma Membrane Na+ -K ATPase 2. HOMOGENIZATION Golgi Apparatus Galactosyl - Disrupts the cell to liberate its Transferase constituents --> resulting suspension contains intact Peroxisomes Catalase, Uric acid organelles known as Oxidase homogenate - Manually operated or motor Cytosol Lactate driven pestle is rotated within Dehydrogenase a glass tube containing the minced fragments of the COMPONENTS TO THE EXPERIMENTAL organ under study and a APPROACH USED IN BIOCHEMISTRY suitable medium Isolation of biomolecules and 3. CENTRIFUGATION organelles Methods: - done at successively greater 1. Salt fractionation speed, each yielding a pellet 2. Chromatography – paper, ion and supernatant exchange, thin layer 3. Gel filtration 3 pellets : 4. Electrophoresis – paper, high - nuclear fraction voltage, agarose - mitochondrial fraction 5. Ultracentrifugation - microsomal fraction - Content of final supernatant Determination of structure of corresponds to the cytosol biomolecules Methods: - fractions are not absolutely pure 1. Elemental analysis organelles; measured with suitable 2. UV, visible, infrared, Nuclear marker enzymes or chemical Magnetic resonance components spectroscopy 3. Use of acid/alkaline hydrolysis to degrade the biomolecules to its basic components 4. Use of enzymes 5. Mass spectrometry 6. Sequencing methods ( for  proteins and nucleic acids) 7. X-ray crystallography Analysis of function and metabolism of biomolecules - Studies maybe done on different levels - whole animal, isolated perfused organ, tissue slice, isolated cell organelle - analysis of biochemical process or metabolic pathway by use of isotopes WATER AND PH THE “POWERS” OF WATER - Can dissolve most organic and WATER IN THE BODY inorganic molecules because of its dipolar structure and ability to form - Total body water is roughly 50 to hydrogen bonds 60% of body weight in adults and 75% of body weight in children - Water is an excellent nucleophile, which enables it to participate in - Because fat has relatively little many chemical reactions water associated with it, obese people tend to have a lower - It exhibits a slight but important tendency to dissociate, enabling it to percentage of body water than thin act either as an acid or base and people, women tend to have a lower playing a role in maintaining pH percentage than men, and older people have a lower percentage than younger people DIELECTRIC CONSTANT - Also called PERMITTIVITY - Approximately 60% of the total body - A measure of the ability of a material to resist the formation of an water is intracellular and 40% electric field within it. extracellular. - Water decreases the force of - The extracellular water includes the attraction between charged particles fluid in plasma (blood after the cells because of its high dielectric have been removed) and interstitial constant - This enables water to dissolve salts water (the fluid in the tissue spaces, lying between cells) - Hexane 1.9 - Ethanol 24.3 - Water 78.5 - Thus, a compound with a high dielectric constant can easily break ionic bonds WATER IS AN EXCELLENT NUCLEOPHILE - A nucleophile forms a chemical bond DEHYDRATION by donating bonding electrons - All molecules or ions with a free pair Dehydration, or loss of water, occurs when of electrons can act as nucleophiles salt and water intake is less than the combined rates of renal plus extrarenal “Dissociation” of water (non-kidney related) volume loss WATER CAN ACT BOTH AS AN ACID (DONATES PROTONS OR HYDROGEN ATOMS) AND AS A BASE (ACCEPTS PROTONS OR HYDROGEN ATOMS) TYPES OF CHEMICAL BONDING 3. Covalent Bonds - Covalent bond is a type of Bonding chemical bond that involves There are two groups of bonds, the the sharing of electron pairs strong bonds called the primary bonds, and between atoms. It occurs the weaker secondary bonds. when two atoms approach each other closely enough so - Primary bonds consist of three types that the electrons from each of bonds: ionic, metallic, and atom are attracted to the covalent nuclei of both atoms, resulting in the formation of a - Secondary bonds are weak bonds stable molecule. existing in substances such as water (hydrogen bond, Van der Waals bond) Secondary Bonds: Primary Bonds: 1. Hydrogen Bonding - A hydrogen bond is the 1. Ionic Bonding attractive force between one - In ionic bonding a element electronegative atom and a gives an electron to an atom hydrogen covalently bonded which needs extra electrons. to another electronegative This causes both atoms to be atom. charged. One has a positive charge (it has more protons - It results from a dipole-dipole force than electrons) and the other with a hydrogen atom bonded to a negative charge. This nitrogen, oxygen, or fluorine causes an attraction between the atoms. - Must be differentiated from a covalent bond, which is simply a - Materials bonded this way sharing of electrons are usually brittle with poor electrical conductivity. 2. Van der Waals Bond - Van der Waals bonds are 2. Metallic Bonds formed from an electrostatic - In metallic bonding, instead charge in adjacent atoms of sharing electrons between two atoms, the electrons in - Present between the outer shells are shared long-chained molecules in among all the atoms in a polymers bonding the chains lattice with all the atoms together positively charged. These atoms are attracted to the - When stretched the bonds negatively charged 'cloud' of break easily causing the electrons. material to deform. - The movement of the free electrons means that metallic bonded materials have good thermal and electrical conduction. Strength of Bonds - The relative strengths of weak acids and weak bases are expressed quantitatively as their dissociation BOND ENERGY EXAMPLE constants (Ka) (GPs) Covalent 1,000 Diamond - pKa is the negative logarithm of Ka - Thus, the lower the pKa, the higher Ionic 30-100 Salt and the dissociation constant Ceramics - The higher the dissociation constant, Metallic 30-150 Metals the greater is the tendency to dissociate or separate into ions Hydrogen 8 Ice NOMENCLATURE Van de 2 Polythene - The name of an undissociated acid Waals usually ends in “ic acid” (e.g., acetoacetic acid) and the name of the dissociated anionic component ELECTROSTATIC INTERACTION ends in “ate” (e.g., acetoacetate). - Interactions between charged groups of ions - Electrostatic interactions between oppositely charged groups within or WHAT HAPPENS AFTER HYDROGEN IS between biomolecules are termed ‘DONATED’ BY AN ACID? SALT BRIDGES DIPROTIC ACID - For acids with more than one HYDROLYSIS BREAKS BONDS dissociable group (molecules with - Nucleophilic attacks by water result carboxyl and amino groups, for in the cleavage of amide, glycoside, example), the dissociation of or ester bonds hydrogen is influenced by changes - This is called HYDROLYSIS in pH DIPROTIC AND TRIPROTIC ACIDS HAVE ACIDS AND BASES MULTIPLE pKs - Acids are proton donors, while bases are proton acceptors - Strong acids like HCl(Hydrochloric Acid) and H2SO4(Sulfuric acid) completely dissociate in strongly acidic solutions with low pH - Weak acids (like acetic acid) dissociate only partially - Strong bases (KOH, NaOH) and weak bases (Calcium hydroxide) also behave the same way in high pH solutions WEAK ELECTROLYTES - The value of Kw when measured at 25°C has been determined to be 1 x 10-14. - Mathematically, if one takes the log of both sides of the equation, remembering that when numbers are multiplied their logs are added, we - Kw is used to represent the equilibrium constant for the ionization of water. It is a special notation of the general representation Kc. - The value of Kw when measured at 25°C has been determined to be 1 x 10-14. DERIVING THE DISSOCIATION CONSTANT OF WATER - Mathematically, if one takes the log - The value for Kw (Dissociation of both sides of the equation, constant of water) comes from remembering that when numbers determining the concentration of are multiplied their logs are added, H3O+ and OH- at 25 degrees we arrive at the following equation. Centigrade from experimentation. - log (1 x 10-14) = log [H3O+] + log - The concentration is 1 x 10-7 for [OH-] H3O+ and OH- - Pure water is 55.56 mol/L - The log (1 x 10-14) = - 14. - Substituting from the dissociation - Thus, - 14 = log[H3O+] + log [OH-] equation, we have: - Multiplying both sides of the equation by -1, we get: - K = [H+][OH-] / [H2O] - K = [1 x 10-7 ] [1 x 10-7 ] / - 14 = - log [H3O+] - log [OH-] [55.56]=1.8 x 10-16 mol / L - Kw = (K)(H2O) = [H+][OH-] - The - log [H3O+] is defined as the - (1.8 x 10-16 mol / L)(55.56 pH, and the - log [OH-] is defined as mol/L) the pOH. Substituting into the - = 10-14 (mol / L)2 equation yields the relationship between pH and pOH of a solution. - Kw equals 10-14 (mol / L)2 for all aqueous solutions, even solutions of - 14 = pH + pOH acids or bases Why pH + pOH = 14 - Kw is used to represent the equilibrium constant for the ionization of water. It is a special notation of the general representation Kc. pH: NEGATIVE LOGARITHM OF Taking the -log of both sides gives us: HYDROGEN ION CONCENTRATION - pH = - log [H+] -log Ka = -log {[A-][H+]/[HA]} - For pure water at 25 ° C, - pH = - log [H+] = - log 10-7 = - (-7) = THE HENDERSON-HASSELBALCH 7.0 EQUATION - Low pH denotes high [H+] and vice The left side is the definition of pKa: versa pKa = -log {[A-][H+]/[HA]} The right side can be re-written using the LOGARITHMIC TABLE log rules: pKa = -log [A-] - log [H+] - (-log [HA]) The second right side term is the definition of pH: pKa = -log [A-] + pH + log [HA] Solving for pH: pH = pKa + log [A-] - log [HA] Using the log rules in reverse gives us the equation as it is normally written: pH = pKa + log [A-]/[HA] Where pKa is the negative logarithm of the pH and pOH acid’s dissociation constant pH - “power of Hydrogen” Remember: The stronger the Acid, the - Measure of the H+ lower is its pk value (hydronium) content in a solution - pH = -log[H+] pOH - 14 = pH + pOH (assuming room temperature [25 degree C] pH scale - neutral: pH = 7 - acidic solution pH < 7 - basic solutions pH > 7 THE HENDERSON-HASSELBALCH EQUATION Derived from the formula for the dissociation constant of an acid, Ka: Ka = [A-][H+]/[HA] APPLICATIONS OF THE HENDERSON Bicarbonate Buffer HASSELBALCH EQUATION - It is the principal extracellular buffer, - The behavior of weak acids and comprising carbonic acid (the proton buffers is described by the donor) and bicarbonate (the proton Henderson Hasselbalch equation acceptor). - The pH of a solution containing a - It functions in the same way as other weak acid is related to its acid conjugate acid-base pairs. However, dissociation constant there are important differences: - Solutions of weak acids and their 1. The base constituent, conjugate bases (or of weak bases bicarbonate ( 3 HCO-) is and their conjugate acids) exhibit regulated by kidneys. BUFFERING, or the tendency of a solution to resist a change in pH 2. The acid component following the addition of a strong (H2CO3) is regulated by acid or strong base pulmonary ventilation. - Important physiologic buffer systems REGULATION OF BLOOD PH include bicarbonate, inorganic Done by maintaining carbon dioxide orthophosphates, and intracellular and bicarbonate ion concentration proteins such as hemoglobin - The normal pH range of arterial blood is from 7.35 to 7.45 - The kidneys and the lungs also play major roles in maintaining pH - A buffer can be created by mixing a weak acid (acetic acid) with its conjugate base (Acetate) - If an acid such as HCl is then added to such a solution, Acetate can neutralize it, in the process being converted to Acetic acid - If a base is added, Acetic acid can neutralize it, in the process being converted to acetate - Maximum buffering capacity: occurs ± 1 pH unit on either side of pKa. - Physiologic buffers include bicarbonate, orthophosphate, and proteins AMINO ACIDS PROTONIC EQUILIBRIA OF AMINO Contain an amino and carboxylic ACIDS groups attaches to the same a-carbon Amino acids exist as zwitterions - a-Carbon of all amino acids - Zwitterions are molecules that except for glycine is chiral or contain an equal number of ionizable asymmetric Chiral groups of opposite charge and carbon-carbon in which all therefore bear no net charge four groups attached are different; confers optical Functional groups present on amino acids activity has different pKa values Specific properties of the individual amino Net charge of an amino acid depends upon acids are dictated by the nature of their R the pH of the surrounding solution groups ISOELECTRIC pH L-a-AMINO ACIDS pH at which an amino acid bears no Absolute configuration of amino net charge and hence does not move in a acids are referenced to Glyceraldehyde direct current electrical field - All amino acids derived from natural proteins are of the - pH midway between pK values on L-configuration either side of the isoelectric species - Only 20 amino acids occur in - Knowledge of isoelectric pH guides proteins from all forms of life in the selection of conditions for electrophoretic Separation - Differ in their R group - Varying the pH of the medium alters SELENOCYSTEINE the charge of the amino acids Commonly referred to as 21st amino acid - Selenium atom replaces the sulfur of GENERAL PROPERTIES OF AMINO its structural analog, cysteine ACIDS Soluble in polar solvents (water, ethanol); - Present in the active site of several insoluble in non-polar solvents (benzene, enzymes eg, thioredoxin reductase, ether) glutathione peroxidase, deiodinase - High melting point - Do not absorb visible light with the exception of the aromatic amino acids Glycine TEST FOR AMINO ACIDS - Smallest amino acid Treatment of a polypeptide with hot - Can fit in regions inaccessible to HCl hydrolyzes the peptide bonds and other amino acids releases free amino acids; reaction with N- hydroxysuccinimidyl carbamate forms AA with aliphatic or aromatic R group fluorescent derivatives that can be - Hydrophobic separated and identified - Occurs in the interior of proteins Ninhydrin AA with charged R group - Also used in detecting amino - Basic and acidic AAs acids; forms a purple product - Role in stabilizing specific protein with -amino acids and yellow conformation by formation of salt with imine groups of proline bonds PEPTIDES -OH of serine and –SH of cysteine Consist of 2 or more amino acid residues - Nucleophile linked by peptide bond - Function as such during enzymatic catalysis Formation of peptides from amino acids is accompanied by a net loss of one positive -OH of serine, tyrosine and threonine and one negative charge per peptide bond - Undergoes phosphorylation which formed regulates enzyme activity Peptides are charged at physiologic pH Imidazole group of histidine because of their terminal carboxyl and - Important in enzyme catalysis amino groups and when present the - pKa of 6.0 allows it to function as charged R group either a base or acid catalyst at neutral pH PEPTIDE BOND FORMATION Peptides written with the - N-terminal residue at the left NON-PROTEIN AMINO ACIDS - C-terminal residue at the right NOMENCLATURE Peptides can be named as: - Seryl–valyl-tyrosyl-cysteine - Ser-Val-Tyr-Cys - SVYC DRAWING A PEPTIDE STRUCTURE Glutathione 1. Draw a zigzag to represent the - Tripeptide (Glu-Cys-Gly) peptide backbone - Participates in the formation of correct disulfide bonds of many 2. The peptide backbone is made up of proteins repeating N, -C, and carbonyl C 3. Add hydrogen to each -carbon and peptide nitrogen, oxygen to carbonyl carbon, and appropriate R group to each -carbon PEPTIDE BOND Exhibit partial double-bond character No freedom of rotation about the bond that connects the carbonyl carbon and the nitrogen of the peptide bond TRH - Pyro-glutamyl-histidyl-prolinamide - Atyphical, N-terminal glutamate is cyclized to pyroglutamate and C-terminal prolylcarboxyl is amidated TEST FOR PEPTIDE BONDS BIURET TEST - Sample treated with alkaline copper sulfate reagent - Produces a violet color in the Aspartame presence of at least two - Artificial sweetener, 200x sweeter peptide bonds than sugar - Dipeptide;L-aspartyl-L-phenylalanine (methyl ester)  AMINO ACIDS CLASSIFICATION OF AMINO ACIDS ACCORDING TO R GROUPS ALIPATHIC SIDE Glycine (G) Alanine (A) Valine (V) Leucine (L) Isoleucine (I) - Gly - Ala - Val - Leu - Ile - Non-Polar - Non-polar - Non-polar - Non-polar - Non-polar - Smallest Amino Acid With OH Group Serine (S) Threonine (T) Tyrosine (Y) - Ser - Tho - Tyr - Non-aromatic hydroxyl - Non-aromatic hydroxyl - Aromatic with hydroxyl group - Polar (Uncharged) - Polar (Uncharged) - Polar (Uncharged)  With Sulfur Atoms Cysteine (C) Methionine (M) - Cys - Met - Polar - Non-polar - Uncharged (Neutral) - Hydrophobic Acidic groups and their Amides Aspartic Acid (D) Asparagine (N) Glutamic Acid (E) Glutamine (Q) - Asp - Asn - Glu - Gln - Polar - Polar - Polar - Polar - Negatively - Uncharged - Negatively - Uncharged Charged (Neutral) Charged (Neutral) - Amide of an acidic Amino acid  Basic Group Arginine (R) Lysine (K) Histidine (H) - Arg - Lys - His - Polar - Polar - Polar - Positively Charged - Positively Charged - Positively Charged - Has Anomeric rings Aromatic Rings Phenylalanine (F) Tryptophan (W) - Phe - Trp - Non-polar - Non-polar - Tyrosin - Histidine  Imino Acid Proline (P) - Pro - Cyclic - Non-polar Proteins - Exhibit partial double-bond character → no freedom of Proteins: are high molecular weight rotation about the bond polypeptides Rotation possible only at: Classifications of Proteins: - Ca (a-carbon) to Co 1. According to components (carbonyl carbon) bond – - Simple: contain only amino psi(y) angle acids - Ca to nitrogen bond – phi (f) - Complex: contain additional angle non-amino acid materials (heme, carbohydrates, etc) - For amino acids other than glycine, most combinations of f and y angles 2. According to overall shape are disallowed because of steric - Globular hindrance Axial ratio < 10 (usually 3 – 4) Ex: Insulin, albumin, globulin - Fibrous Axial ratio > 10 Ex: keratin, collagen, fibrin 3. According to function Catalytic role: enzymes Contraction: actin, myosin Gene regulation: histone, repressor protein Hormonal role: insulin Protection: immunoglobulin, interferon Regulatory role: calmodulin Structural: collagen, keratin Transport: hemoglobin, albumin, lipoprotein BONDS THAT STABILIZE PROTEINS COVALENT BONDS 1. Peptide bonds - Bond that connects the carbonyl carbon and the amino nitrogen 2. Disulfide bonds NON-COVALENT BONDS - Link two portions of 1. Hydrogen bonds polypeptide chain through a Between the side chains of cystine residue amino acids, between H and O atoms of the peptide - Resistant to conditions for bonds, between polar denaturation residues on the surface of the proteins and water - Cleaved by performic acid and b-mercaptoethanol *Performic acid – oxidizes S – S bonds *b-Mercaptoethanol – reduces S – S bonds 2. Hydrophobic interactions - Between non-polar side chains of amino acids - Contribute stability to interior of proteins - Not a true bond  B. Repulsive force - when two atoms come so close that their electron orbitals overlap 3. Electrostatic bonds Between oppositely charged groups in the side chain of amino acids, between N-terminal and C-terminal residues, and other oppositely charged groups **combined 4. Van der Waals interaction - Involves neutral atoms - Extremely weak, act only over extremely distances Much weaker than hydrogen bonds A. Attractive forces - involves interaction between induced dipoles formed by momentary fluctuations in the electron distribution in nearby atoms FOUR ORDERS OF PROTEIN - Distance/turn = 0.54 STRUCTURE nm 1. Primary structure - Sequence of amino acids - Stabilized by H-bonds - Determines the eventual between H atom of N and O secondary, tertiary and of carbonyl of residue 4th in quaternary structure of a line behind protein - All peptide bonds participate Determination of Primary Structure in H bonding A. Sanger Sequencing - Sequenced insulin - Lowest energy; most stable - Used 1-fluoro-2,4 conformation R-handed helix dinitrobenzene more stable than L-handed helix B. Mass Spectrometry - Based on difference in the - AA that disrupt a-helix: mass-to-charge (m/z) ratio of proline and AA with charged ionized atoms or molecules or bulky R groups - Not only determines amino acid sequence but also identifies post-translational modification 2. Secondary structure - Regular arrangement of amino acids located near to each other in the linear sequence Types of Secondary Structure A. a-HELIX **From the Top - Arise when series of amino acyl residues adopt similar phi and psi angles - 57o for phi angle - 47o for psi angle - Characteristics: - 3.6 residues/turn - Spacing/aa residues = 0.15 nm B. b-PLEATED SHEETS - Connects ends of two - Types: adjacent strands of - Anti-parallel: when anti-parallel b-sheets adjacent poly-peptide chains run in opposite - Makes tight 180o turns direction involving 4 amino acids - Parallel: when - Occurs primarily at protein polypeptide chain run surfaces in same direction - Often contains proline and - Stabilized by H-bonds glycine between peptides far removed from on another in a primary structural sense RANDOM COILS - Disordered conformation of denatured proteins - Not a secondary structure SUPERSECONDARY STRUCTURES - Structural motifs that are combinations of several secondary structures - eg. Helix-loop-helix, Greek key, ab Barrel, bb Barrel C. b-BENDS  4. Quaternary structure - Aggregation of 2 or more polypeptide chains 3. Tertiary structure - Assembly of secondary structural units into larger functional units (domains) **LEVELS OF PROTEIN STRUCTURE - Tertiary structures are stabilized five ways: - Covalent Bonds - Hydrogen Bonds - Salt Bridges - Hydrophobic Interactions - Metal Ion Coordination DENATURATION - Disruption of native conformation - For polymeric proteins, subunits assemble to form 4o structure - Disruption of weak forces responsible for maintaining 2o, 3o - Further series of slight and 4o structure but not covalent conformational adjustments yield bonds native protein structure - Loss of biologic activity STAGES IN PROTEIN FOLDING - Becomes less soluble - Denaturating agents: strong acid and base, heat, ionic detergent, urea, guanidine, heavy metals, organic solvents PROTEIN FOLDING - Proteins spontaneously fold into their native conformation under physiologic conditions - Dictated by the protein’s primary structure STAGES IN PROTEIN FOLDING - Formation of short stretches of 2o structure – a-helix, b-sheets, b-bends FOLDING ACCESORY PROTEINS 1. Protein Disulfide Isomerase - Growth of the short stretches leading - Catalyzes the random to formation of domains cleavage and reformation of a protein disulfide bonds - Folded domains coalesce to form thereby interchanging them “molten globule” (entity that has as the protein progressively extensive 2o structure but attains a thermodynamically disordered 3o structure more favorable conformation STAGES IN PROTEIN FOLDING - Series of relatively small conformational adjustments convert “molten globule” to make compact 3o structure – native polypeptide structure  2. Prolyl cis-trans isomerase Consist of: - ~10% of X – Pro peptide - Hsp 60 (GroEL in E.coli and Cpn 60 bonds of mature proteins in chloroplast) have cis conformation - Hsp 10 (GroES in E.coli and Cpn10 - Catalyzes the otherwise slow in chloroplast inter-conversion of X – Pro peptide bonds between cis *Heat shock protein and trans conformation - rate of synthesis increases at thereby facilitating the folding elevated temperature; reverses the process denaturation and aggregation of proteins 3. Molecular chaperones - Large multi-subunit proteins that accelerate the folding process by providing a protected environment where - Proteins fold to achieve the optimum polypeptides fold into their conformation it requires to perform native conformations and its function form quaternary structure PROTEIN STRUCTURE-FUNCTION - Inhibit inappropriate RELATIONSHIP interactions between Proteins have unique conformations potentially complementary suited for their function surfaces and disrupts unsuitable liaisons so as to CLASSIFICATION OF PROTEINS facilitate more favorable 1. FIBROUS associations - Elongated structure; has repeating structural motif CHAPERONINS Eg. a-keratin, silk fibroin, - Cage-like proteins found in bacteria, collagen mitochondria, chloroplast and eukaryotes 2. GLOBULAR - Spherical structure; polypeptide chain folded back on themselves Eg. hemoglobin, myoglobin FIBROUS PROTIENS - Twisting occurs so that side 1. a-KERATIN chain residues falling on - Constituent of hair, wool, same side of a helix do not horns and outer skin; part of lie along a line parallel to the the intermediate filament of helix axis (to optimize cytoskeleton packing among amino acid side chain residues between - Two R handed a-helices helices) aggregate side by side to form long cables with a - Springiness of hair and wool L-handed twist fiber results from tendency of a-helical cables to untwist - Surface between the two when stretched and spring helices touch are made up of back when force is removed hydrophobic amino acids – Ala, Val, Leu, Ile, Met, Phe - Keratin contains a high percentage (about 11%) of cysteine. The SH groups in two cysteine residues can be oxidized to form a disulfide bridge that stabilizes the tertiary structure. 2. SILK FIBROIN 3. COLLAGEN - Composed of stacked - Found in tendon, skin, teeth, anti-parallel b-sheets bones, cornea - Consist largely of glycine, - Has Gly-X-Y sequence; Y is serine, and alanine in which usually proline or every alternate residue is hydroxyproline glycine - Only Gly can be accommodated at the - One side of b-sheet will be all very tight junctions glycine or all alanine so that between individual in a stacked arrangement, chains glycine or alanine surfaces - Pro permits sharp interlock with each other twisting of helix - Tropocollagen: fundamental unit of collagen - Consist of 3 polypeptide chains in R-handed triple helix stabilized by H bonds - Each polypeptide chain forms a L-handed helix having 3 residues/turn - Stabilized by extensive hydrogen bonding and Van der Waals - Undergo aldol condensation interactions and cross-linking conferring great tensile strength - Mechanically rigid: resist stretching due to extended composition of b-sheets and interlocking of side chains between sheets