Peptides The peptide bond formed between the carboxyl group of one amino acid and the amino

amino group of the next one an amide bond formed by elimination of the elements of H2O a resonance hybrid of 2 structures

. .. O.
C

C N

.. C
H

+C C N C H

peptide bond is planar, rigid and has partial double bond character stereochemical constraint plays a role in determining folding in protein backbone - N-H group of peptide linkage has no significant tendency to ionize or protonate (between ph 1 14)

Physiologically active peptides glutathione enkephalins oxytocin vasopressin gramicidin S tyrosidine A Acid base properties

Levels of Protein Structure Many different conformations are possible for a protein. The native conformation is that conformation under normal biological conditions of temperature and pH in which the protein is biologically active. Primary Structure the linear sequence of amino acids in a protein determines the higher orders of structure and biological activity of the protein includes covalent interactions in the linear chain and stabilized by resonance (via the peptide bond) results of amino acid substitutions may range from negligible effects to complete loss of activity, depending on the protein and the nature of the altered residue changes in amino acids that do not dramatically alter the character of the residue are called conservative replacements

Secondary Structure regularly repeating conformation of the peptide chain considers only the peptide chain backbone (and not the side chains) structures formed by non-covalent bonding between sections of the same chain or adjacent polypeptide chains. principal form of non-covalent bonding is the hydrogen-bonding between the oxygen atom of the carbonyl group of one amide group with a hydrogen attached to the nitrogen of another amide group further along the chain also stabilized by hydrophobic interactions The -helix and -pleated sheet are periodic structures; their features repeat at regular intervals. the -helix a rod-like, regular coiled conformation, involving only one polypeptide chain stabilized by H-bonds between an N-H in the polypeptide chain and a C=O group at a position 4 residues away in the same chain contains 3.6 amino acid residues per turn of the helix the side chains stick out factors that disrupt the helix: (a) the presence of proline creates a bend in the backbone, (b) strong electrostatic repulsion due to the proximity of several charged groups of the same sign, (c) crowding caused by the proximity of several bulky side chains (side chains lie outside the helix)

the -pleated sheet side-by-side polypeptides connected by H-bonds where the polypeptide chain is almost fully extended rather than tightly coiled. formed by polypeptides with repeating sequences of amino acids with compact side chains can involve one or more polypeptide chains: H-bonds can be formed between different parts of a single chain that is doubled back on itself ( intrachain bonds) or between chains H-bonds are perpendicular to the direction of the protein chain, not parallel to it. if the peptide chains run in the same direction (aligned in terms of their Nterminal and C-terminal ends) a parallel pleated sheet is formed; when alternating chains run in opposite directions, an antiparallel pleated sheet is formed.

Irregularities in Regular Structures A -bulge is a common nonrepetitive irregularity found in antiparalllel -pleated sheet. This occurs between 2 normal -structure H-bonds. other helical structures found in shorter stretches than with the -helix) can break up the regular nature of the -helix A reverse turn marks the point at which the polypeptide chain changes direction and for steric reasons, glycine is most often encountered; proline is also often encountered in such turns

Supersecondary structures two parallel -pleated sheets connected by an -helix unit two successive -helices separated by a loop or non-helical segment become enmeshed because of compatible side chains -meander two antiparallel -pleated sheets connected by polar amino acids and glycines to effect an abrupt change in the direction of the polypeptide chain called reverse or -turns Greek key antiparallel -pleated sheet doubles back on itself A motif is a repetitive secondary structure. Protein sequences that allow for a -meander or Greek key can often be found arranged into a -barrel.

Tertiary Structure Types of Protein Conformations Fibrous elongated, often very large molecules, often composed of 2 or more polypeptide chains, generally water-insoluble Globular have a more compact structure due to a highly ordered pattern of folding, bending and twisting; may consist of a single polypeptide chain or an aggregate of 2 or more chains which may be identical or different; more soluble in water The tertiary structure: 3D arrangement of all the atoms in the molecule for fibrous proteins, the only aspect of tertiary structure not specified by the secondary structure is the arrangement of atoms of the side chains in globular proteins, interactions between side chains play an important role and amino acid residues that are distant from each other in the primary structure come into close proximity forces involved in protein folding: hydrogen-bonding, hydrophobic interactions, electrostatic interactions, disulfide bonds globular proteins are intact most water molecules are excluded from the protein interior large globular proteins (those with more than 200 amino acid residues) often contain several compact units called domains (structurally independent segments that have specific functions). Domains found in several proteins: EF hand - consists of a helix-loop-helix configuration; binds specifically to Ca 2+ Zinc finger common in DNA-binding proteins; cysteine residues provide the binding site for Zn ions Leucine zipper knobs on the zipper represent Leucine side chains; also DNAbinding domain the primary structure contains all information necessary to produce the tertiary structure; special proteins called chaperones aid in the correct and timely folding of many proteins

Quaternary Structure a property of oligomeric proteins (proteins consisting of more than one polypeptide chain); each chain is called a subunit or protomer the chains interact with one another non-covalently (hydrogen bonds, electrostatic attractions and hydrophobic interactions) many multi-subunit proteins exhibit allosteric properties (small changes in structure at one site may cause drastic changes in properties at a distant site)

Some Examples of Fibrous Proteins Alpha Keratins relatively rich in cysteine residues and disulfide bridges proteins of horn and nails are hard and brittle and have up to 22% Cys; those of hair, skin and wool contain 14% cysteine and are softer, more flexible and elastic In the alpha keratins of hair and wool, 3 or 7 helices may be coiled around each other to form ropes held together by S S linkages. These are known as protofibrils. The protofibrils are in turn coiled into a higher order, cable- like structures called microfibrils. Alpha Keratin is a coiled-coil. It is strong, inextensible, insoluble and chemically inert. The consensus amino acid sequence is a repeating heptamer of (-a-b-c-d-e-f-g-)n where residues a and d are non-polar. The two alpha helicies coil, rather than form a ladder, because each alpha helix turns 700 degrees from one a residue to the next. Beta Keratins Silk Fibroin is strong, inextensible, insoluble and chemically inert; extension resisted by full strength of covalent bonds in silk fibroin, antiparallel beta pleated sheets contain sequences of (-Ser Gly Ala Gly- )n interrupted by regions containing bulkier residues. In sum the fiber is composed of microcrystalline arrays alternating with amorphous regions. fiber strengths result from extensive H bonding of adjacent chains and the cumulative effect of van der Waals interactions among stretched chains Collagen limited to polypeptides with extensive sequences of ( -Gly X Pro -)n or ( - Gly X OH Pro -)n consist of 3 helices H bonded to each other to form a right handed superhelix (only glycine can fit into the interior portions of the superhelix) structure is responsible for the tensile strength of connective tissue proteins (collagen is 1/3 of total body proteins, it is the most abundant protein in vertebrates). Collagen gives strength to bones, teeth, cartilage, tendon and the fibrous matrices of skin and blood vessels. fibrils are arranged in different ways depending on the biological function of connective tissue bone collagen plus calcium phosphate polymer tendons rope-like fiber of high tensile strength skin collagen fibers loosely woven and can expand blood vessels fibers arranged in elastic helical networks eye well-ordered, nearly crystalline Cross linking via lysine side chains also contribute to toughness of collagen; some Lys are oxidized to aldehyde derivatives which react further via condensation and dehydration to form cross links. This process continues

through life and accumulating cross links make collagen less elastic and more brittle

Some Examples of Globular Proteins

Myoglobin in muscle, stores oxygen and enhances its rate of diffusion through the cell a small globular protein with a single polypeptide chain (backbone consists of 8 helical segments) contains a heme group (Fe porphyrin ring) which is the oxygen binding site of myoglobin

the heme group

Myoglobin

Hemoglobin

Hemoglobin constitutes about 90% of the total protein in erythrocytes; O 2 carrier oligomeric protein (4 sub-units, ,) both and chains are similar to myglobin, many amino acids are homologous (the same amino acids in the same positions) contains 4 heme groups, binds 4 O 2 molecules; heme group similar to that in myoglobin exhibits allosteric properties (involve interactions between spatially distant sites) arising from sub-unit interactions binds O2 reversibly (like myoglobin) - O2 binding exhibits positive cooperativity (when one O2 is bound, it becomes easier for the next to bind - Both H+ and CO2 affect affinity of hemoglobin for O2 Bohr effect the affinity of hemoglobin for O2 depends on pH H+ + HbO2 + H + CO2 O2 + Hb CO2 Lungs Higher pH than actively metabolizing tissue Hemoglobin binds O2 Hemoglobin releases H+ Actively metabolizing muscle Lower pH due to production of H+ Hemoglobin releases O2 Hemoglobin binds H+

Hemoglobin in blood is also bound to 2,3-bisphosphoglycerate (electrostatic interactions between negative charges on BPG and positive charges on protein). Fetal hemoglobin binds less strongly to BPG and has greater affinity for O 2 than does maternal hemoglobin. This allows for efficient transfer of O 2 from mother to fetus.

Oxygen binding curves for Myoglobin and Hemoglobin

Effect of pH on O2 - binding to hemoglobin

Determination of the Primary Structure of Proteins 1. Separate individual chains if protein contains more than one polypeptide chain (deduce from number of N-terminal residues) 2. Break all S-S linkages (use reducing agents then alkylate) 3. Subject a sample of each polypeptide chain to hydrolysis and determine amino acid composition 4. Determine N-terminal and C-terminal amino acid residues of each polypeptide chain N-terminal: by Edman degradation, DNFB or DANSYL chloride C-terminal: by cleavage reaction with carboxypeptidase 5. Cleave the intact polypeptide chain into smaller peptides by enzymatic or chemical cleavage Trypsin: cleaves at the C-side of Lys, Arg (+ charged R groups) Chymotrypsin: cleaves at the C-side of Phe, Trp, Tyr (aromatic amino acids) Thermolysin: cleaves at the N-side of Leu, Ile, Val CNBr (cyanogens bromide): cleaves internal methionine residues 6. Separate the peptide fragments (in step 5) and determine amino acid composition and sequence (by Edmans degradation) 7. Order the fragments by repeating steps 4 and 5 and using a cleavage procedure of different specificity to generate overlap peptides

Exercises 1. Polypeptide A has the following amino acid sequence: Val-Ala-Cys-Glu-Glu-Phe-Val-Met-Tyr-Cys-Gly-Trp-Gly-Met-Phe-Gly (a) list the peptide formed when polypeptide A is treated with chymotrypsin (b) list the peptides formed when polypeptide A is treated with thermolysin

2.

Polypeptide B has the following amino acid composition: Cys, Ala, Asp, Gly, Ile2, Leu, Tyr, Val, , Phe, Lys2, Arg, Ser, Met, His Treatment with trypsin and subsequent Edmans degradation gave the following peptides: Asp-Gly-Ile-Phe-Ile; Leu-Met-His-Tyr-Lys Ser-Val-Cys-Ala-Lys; Arg Treatment with thermolysin followed by Edmans degradation gave: Ile-Phe; Val-Cys-Ala-Lys-Asp-Gly Ile Leu-Met-His-Tyr-Lys-Arg-Ser Deduce the sequence of the polypeptide