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Protein structure and Function Objectives: Structure of amino acids as a building unit of proteins. Acidic and basic properties of amino acids. Levels of protein structure Protein misfolding. Protein structure and Function Proteins are the most abundant and functionally diverse molecules 1. function: enzymes, hormones, protein in muscle, receptors, Hb & Mg 2. Structure: hair, nail, bone, skin…. etc Amino acid (AA) structure: AA are building blocks of proteins Only 20 AA are found in mammalian proteins & much more are found in nature (<300) General Structure of amino acid Classification of AA AA with Nopolar side chains AA with polar uncharged AA Acidic AA Basic AA AA with imino group You should know names, structures, three letters & one letter AA with nonpolar side chains AA with nonpolar side chains Each of these AA has a non polar side chain that does not give off proton or participate in hydrogen or ionic bonds The non polar R group fill up the anterior of the folded protein and give it its three-dimensional shape. In proteins that are located in a hydrophobic environment, such as membrane, the non-polar R group are found on the outside surface of the protein, interacting with the lipid content. This plays an important role in stabilizing protein strucure. AA with Uncharged Polar Side Chains AA with Uncharged Polar Side Chains These AA have zero net charge at neutral pH. Serine and threonine: Each contains a polar hydroxyl group that can participate in hydrogen bond formation. Additionally this polar hydroxyl group can serve as a site of attachment of structure such as phosphate group or an important component of active site of many enzymes. Asparagine and glutamine: Each contain carbonyle group and amide group can participate in hydrogen bond. Moreover it can serve as asite of attachment of oligosaccharide chains in glycoproteins. As well as serine Tyrosine: has phenolic group that carries negative charge at pH above its PKª ( pH = 10.5), so it is not hydrophobic at this pH range. AA with Uncharged Polar Side Chains Cysteine: In proteins, SH group (sulfhydry) of two cysteine become oxidized to form dimer Cystine, which contains covalent cross-link called disulfide bond (S-S). AA with acidic side chain They are proton donors. At physiological pH, these AA are negatively charged ( - ve are present on the side chain. -Aspartic acid -Glutamic acid AA with basic side chain They are proton acceptors. At physiological pH, these AA are positively charged ( + ve are present on the side chain. -Histidine -Arginine -Lysine Proline Side chain of proline and its α -amino group form a ring structure, so proline differ from other amino acids. It contains Imino group Unique geometry of proline contributes to the formation fibrous structure of collagen and interrupts of the α -helix found in globular proteins Metabolic classification of amino acids Ketogenic or glucogenic amino acids. 1- Ketogenic amino acids whose catabolism yields either acetoacetate or one of its precursors. 2- Glucogenic amino acids whose catabolism yields pyruvate or one of the intermediate of citric acid cycle. Biological classification of Amino Acids Essential or nonessential amino acids. Optical Properties of AA α -carbon of AA is attached to four different groups & therefore is optically active. They are called Enantiomers or Stereoisomers All AA are optically active except Gly Optical Properties of AA Amino acids have asymetric center on α-carbon atom so they exist in two forms L & D forms. All amino acids found in mammalian proteins are L- form, however Dform is found in bacterial cells. Optical Properties of AA O H2N CH C OH R Proteins are made of amino acids - a-amino acids - only L-isomers found Acidic and Basic Properties of AA Acids: Compound that donate protons. Bases: Compound that accept protons. Strong acids: Ex, Hcl which dissociated completely. Weak acids: Ex, Acetic acid which dissociate only to a limited extend. Buffer: It is a solution that resists change in pH following the addition of an acid or base. It can creat by mixing a week acid and its conjugate base. Acidic and Basic Properties of AA AA in aqueous solution contain weekly acidic α-carboxyl & α-amino groups in addition to acidic & basic side chains AA Henderson-Hasselbalch equation: HA H+ + A - Weak acid conjugate base Ka= [H+] [A-] [HA] Acidic and Basic Properties of AA Taking the logarithmic of both sides of the equation. Multiplying both sides of equation by -1 Substitution pH= - log [H+] , and pKa = -log ka we obtain HendersonHasselbalch equation Acidic and Basic Properties of AA pH = pKa + log [A-] [HA] The larger Ka & the smaller pKa ,the stronger the acid & vice versa Buffers: solution that resists change in pH following addition of acid or base. Maximum buffering capacity occurs at pH=pKa & effective for ± 1 pH unit of pKa. At pH values less than pKa, protonated acid form is predominant. At pH values greater than pKa , deprotonated base form is predominant in solution. Titration of Acetic Acid A solution containing acetic acid (HA=CH3CooH),and acetate(A- = CH3Coo-) with a PKa of 3.8 resists a change in pH from 3.8 to 5.8 with maximum buffering capacity at pH 4.8. At pH< PKa the protenated acid form is predominant (CH3CooH) At pH> PKa the deprotenated base form is predominant (CH3Coo-) Titration of Amino Acid Zwitterion is isoelectric (pI) form with zero charge pI is the average of pK1 +pK2 /2 Isoelectric point: pH at which an amino acid is electrically neutral, that is the sum of positive charge equal the sum of the negative charges. Titration Curve of Alanine At low pH both (CooH & NH2) are protenated. As pH of the solution raised (CooH) dissociated by donating a proton in the medium forming hydroxylate group (Coo-) so the molecule is dipolar form called Zwitter ion or Isoelectric forms Application of Henderson-Hasselbalch Equation Henderson-Hasselbalch Equation can be used to calculate how the pH of the physiological solution responds to change in the concentration of week acids and /or salt form. Application of HendersonHasselbalch Equation Bicarbonate as a buffer: pH=pKa+ log [HCO3-] [H2CO3] ↑ bicarbonate ↑ pH ↑ CO2 as in pulmonary obstruction pH Drug Absorption Drugs are either weak acid or weak bases & the uncharged form is more permeable through Membranes. Acid: HA H+ + ABase: BH+ B + H+ Drug Absorption however, acid drugs permeate more in low pH whereas basic drugs permeate more in high pH Example: Aspirin Buffering occurs within ± pH unit of the pka where [A-]=[HA] Activity 1 Classify each of the 20 amino acids according to the side chain on the a carbon as non-polar, polar, sulfurcontaining, basic, acidic, or amide derivative Primary Structure of Proteins How is protein formed? By peptide bond Character of peptide bond: Amide bond between α-COOH & α-NH2 Not broken by heating or urea Can be broken by strong acids or base at high temp. or by proteolytic enzymes Peptide bond AA are joined covalently by peptide bond which are amide linkage between α-carboxyl group of one amino acid and α-amino group of another. Partial double bond Rigid & planar Uncharged but polar -NH & -C=O groups are involved in hydrogen bonding Amino acids can be linked by peptide bonds Peptide bond - condensation reaction (loss of one H2O) -Several amino acids can be linked, forming a polypeptide chain - backbone: -NH2, a-Carbon, -COOH ; side chains protrude from backbone - Convention: amino terminus taken as beginning of polypeptide: N C Side chains Peptide backbone Side chains Peptide bonds may be cis or trans configured The planar arrangement of the atoms in the peptide bond (which is required for resonance) can be realised in two ways: Trans: Carbonyl-O and amide-H on different sides of the peptide group Cis: Carbonyl-O and amide-H on the same side of the peptide group Usually, the trans configuration is strongly favoured, since there is no steric hindrance H C–N O O H C–N Peptides – polarity N C N-terminus (amino end) C-terminus (carboxyl end) Naming the peptide Free NH2 on left is N-terminal & free COOH on the right is C-terminal All proteins are read from N- to C- terminal end of peptide Polypeptide is linkage of many AA via peptide bonds Each AA in a polypeptide is called a residue or moiety Ex: val-gly-leu is called valylglycylleucine Levels of protein Structure Primary structure: sequence of AA in a polypeptide chain. Important in genetic disease. Secondary structure: regular arrangements of AA in linear sequence (Ex.α-helix ,β-pleated sheet & B-bend).It describe the geometrical arrangment of polypeptide bond around one axis. Tertiary structure: folding of protein domains of a single polypeptide chain in three dimensional structure. Quaternary: more than one polypeptide chain. Secondary Structure of Protein 1. α-helix: Spiral structure Coiled polypeptide backbone core with the side chains of AA extending outward from central axis to avoid sterric hindrous Stability of α-helix is by hydrogen bond formation between -C=O----HN-( Carbonyl O2 and amide H2 Secondary Structure of Protein Each turn of α-helix contains 3:6 of AA AAs that disrupt α-helix are: Pro (insert a kink) Charged AA e.g glu,asp,his,lys, arg (form ionic bonds) Bulk side chain AAs e.g trp,val,Ile Peptide backbone N-C-C-N Secondary Structure of Protein β-Sheet All of peptide bonds are involved in H.B and, therefore, is fully extended. Appears pleated & as arrows and can be formed from two or more separate polypeptide chains. H.B is perpendicular to polypeptide backbone & called interchain bonds of separate polypeptide chains or intrachain of a single polypeptide chain Types of β-Sheet Pararllel & antiparallel Compare between α-helix & β-sheet Secondary Structure of Protein 3. B-Bends B-bends (reverse turns) B-bends reverse the direction of a polypeptide chain helping it form a compact globular shape Found on surface of protein molecule Often include charged residues Generally of four AAs one is pro that form kink and gly Stabilized by H.B & ionic bonds 4. Nonrepetitive secondary Half of globular protein is organized as repetitive structure such as α-helix and β-sheet, the remainder is formed as loop or coil conformation called nonrepetitive secondary structure Nonrepetitive secondary structure are not “random”but have less regular structure than α-helix, β-sheet & Bbends Supersecondary Strucure (Motifs) Secodary structures, such as α-helix, βsheet, nonrepetitive & B-bend, are elements to form the core region (interior of protein mol) and at the surface of protein mol loop regions (Bbends) are the connectors to the core forming the motifs Tertiary Structure Folding of basic units (domain) to form a final arrangement of a single polypeptide chain (monomeric protein) Domain is a functional three dimensional structure units of a polypeptide Polypeptide chains > 200 AAs consist of 2 or more domains Core of a domain is built from different motifs (supersecondary structure) Interactions stabilizing Tertiary Structure Interactions between side chains of AAs determine the way a polypeptide fold to form the compact structure Types of interactions that form tertiary structure: 1. Disulfide bond, covalent strong bond 2. Hydrophobic bond, nonpolar side chains 3. Hydrogen bond, between N or O & H 4. Ionic bond, between - & + groups Interactions stabilizing Tertiary Structure Protein folding: Interaction between the side chains of AA determine how long polypeptide chain folds into the intricate three-dimensional shape of the functional protein. As a peptide folds, its AA side chains are attracted and repulsed according to their chemicals properties. Protein Folding Trial & error of side chain interactions seek the configurations of a protein with a low energy state. Chaperones in Protein Folding Chaperones are specialized gp of proteins required for proper folding of proteins Chaperones also called “heat shock” proteins Chaperones interact with polypeptide at various stages during folding process Functions of Chaperones Act as unfold unit until synthesis is finished Act as catalysts in final stages of folding Protect proteins as they fold Quaternary Structure of Protein Proteins that consist of more than one polypeptide chain (multimeric) If a protein consists of two subunits, protein is dimeric. If three, protein is trimeric Subunits are connected by noncovalent interactions (H.B, ionic & hydrophobic) Subunits may work independently or cooperatively (Hb) Denaturation of Proteins Unfolding & disorganization of proteins' secondary and tertiary structures without hydrolysis of peptide bonds Denaturing agents are: heat, organic solvents, mechanical mixing, acids & bases, detergents & heavy metals (Pb, Hg) Denatured proteins are insoluble & precipitated from solution Protein Misfolding Misfolding of proteins may occurs spontaneously or be caused by a mutation in a particular gene, which then produced an alter protein. In addition, some normal proteins can after abnormal proteolytic cleavage, take on a unique conformational state that leads to the formation of long , fibrillar protein assemblies consists of β-pleated sheets Accumulation of these spontaneously aggregating proteins called amyloids Protein Misfolding If proteins are misfolded (improper folding) & not degraded, these proteins may be deposited & cause diseases called amyloidoses Alzheimer disease is a degenerative disease caused by accumulation of misfolded proteins (amyloid plaque) Alzheimer disease: normal proteins after abnormal chemicals processing , take on unique conformational state that leads to formation of neurotoxin amyloid proteins assemblies consisting of β-pleated sheets. Protein Misfolding Prion disease: It has been strongly implicated as the causative agent of transmissible spongiform encephalopathies ( TSEs) Creutzfeldt-Jacob disease in humans Mad caw disease in cattle. In TSEs, the infective agent is an altered version of a normal prion protein that act as template for converting normal protein to the pathogenic conformation. Activity 2 Define primary, secondary and tertiary structure of proteins, mention the bonds that stabilize each structure.