Invariant residue-a residue that is always conserved. It is assumed that these residues are essential to the structure or function of the protein.

Transcription

1 Chapter 6 The amino acid side chains have polar and nonpolar properties, and the relative hydrophobicity of the amino acid side chains is critical for the folding and stability of a protein. The more hydrophobic amino acids will be found on the interior of the protein (away from water), and the more hydrophilic amino acids will be on the exterior of the protein (in contact with water). Levels of Protein Structure: 1) Primary Structure- The amino acid sequence of the protein (coded for by the DNA of the gene). This determines the protein s structure, mechanism of action, and relationship to other proteins. Example: Insulin-produced in pancreatic islet cells as a single chain precursor proinsulin. 86 amino acids and 3 disulfide bonds Proinsulin Insulin cleave at and to get two chains (A and B) which are joined together by a disulfide bond. The amino acid sequence is very homologous between species except at 4 residues-which apparently do not affect biological properties. Comparison of primary structure is used to predict similarity in structure and function between proteins (homologous-highly aligned sequences). 2 types of substitutions: 1) conservative-substitute an amino acid with one of similar polarity Val Ile 2) nonconservative- substitute an amino acid with one of dramatically different polarity Val Arg Both of these types of substitution may severely affect protein function or the substitution may not be harmful to protein function. Invariant residue-a residue that is always conserved. It is assumed that these residues are essential to the structure or function of the protein. Clinical- Sickle Cell Anemia A single amino acid is altered in the hemoglobin protein. Normal hemoglobin HbA Abnormal hemoglobin HbS E V The mutation leads to the polymerization of hemoglobin molecules and causes them to precipitate within the red blood cell. The red blood cells become sickle shaped and can clog capillaries. Must be homozygous for HbS to exhibit the disease. 2) Secondary Structure- The local 3-D folding of the protein. -NH- C- CO- NH- Can only rotate around φ (phi) and ψ (psi) φ ψ When all φ bonds are equal and all ψ bonds are equal-leads to regular secondary structures (a-helix and the β structure) a-helix: Helical structures in general are characterized by the number of amino acids per turn of helix (n) and the distance between the turns (d). The a-helix has an n=3.6. The a-helix is a very stable structure because can form H-bonds between the carbonyl of the backbone and the amine group in the backbone four residues further up the chain. All the H-bonds lie parallel to the helix axis. In addition, the amino acid side chains are pointing toward the outside of the helix. This will minimize repulsions, but each side chain is still in contact with the amino acid side chains 3 and 4 residues away in the sequence. Will

2 destabilize the helix structure if these amino acids in contact have the same charge or if have a branched side chain (V, I). Proline is an a-helix breaker due to its cyclic structure which fixes the value of φ. β-sheet: Stabilized by local, cooperative formation of H-bonds along the peptide backbone (note: these H- bonds are interstrand). β-sheets consist of β-strands H-bonded to a similar strand in either parallel or antiparallel direction. The H-bonds are close in the parallel structure (0.325 nm) than in the antiparallel structure (0.347 nm). The amino acid side chains are oriented perpendicular to the plane of the sheet. The antiparallel sheets usually arrange all of their hydrophobic residues on one side of the sheet. Antiparallel pleated sheets β-turns The peptide chain forms a tight loop with the carbonyl oxygen of one residue H-bonded to the amide proton of the residue three positions down the chain. Certain amino acids, such as P and G, occur frequently in β-turn sequences. The cyclic structure and fixed φ angle of praline facilitates the formation of a β-turn. 3) Tertiary Structure- The overall 3-D folding of the protein. General Rules of Folded Proteins: 1) hydrophobic side chains are in the interior of structure, away from water surface. 2) Ionized side chains are on the surface of the protein where they are stabilized by water 3) Polar side chains are on surface in contact with water, but 4) Can have buried water molecules in the interior of the protein A long polypeptide strand folds into domains: each domain typically contains continuous amino acids The domains are separated by a section of polypeptide chain or a cleft of less dense tertiary structure. Many enzymes have the active site within a cleft site. Each domain may have a separate function (see Ricin in protein examples). The 3-D structure consists of secondary structures. For example: 1) can have all a-helices joined together to form a globular shape (myoglobin), 2) can contain both a-helices and β-sheets, 3) can have all β-structures. 4) Quaternary Structure- Arrangement of polypeptide chains in a multichain structure. Each protein subunit must be held together noncovalently. Most proteins do not form multi-chain structures, so they do not have quaternary structure. Two overall types of Proteins: 1) Globular Proteins- Named for their approximately spherical shape. Globular proteins exist in an enormous variety of 3-D structures. Proteins are dynamic structures. Conformational changes involve motions of groups of atoms (amino acids) or even whole sections of proteins (distance can be as large as 1nm). These motions occur in response to specific stimuli or arise from specific reactions within the protein. The binding of ligands to the protein can induce conformational changes (important in enzyme catalysis and regulation, receptor response to hormones, etc.)

3 2) Nonglobular Proteins- low water solubility Includes fibrous proteins, lipoproteins, and glycoproteins. A) Fibrous Proteins- An example is Collagen. Collagen has a high percentage of Gly, Pro, 4-hydroxyproline, and 5-hydroxylysine (figure 5.17). In collagen, Gly occurs at every third residue, and Pro or hydroxyproline occurs at every third residue to get repeating units of: Gly-Pro-Y or Gly-X-HyPro This repeat allows for the formation of 3 separate helices with n=3 (not a-helices!!). The three helical chains are wound around each other to form a superhelical structure. The strands are able to intertwine because Gly has a low steric bulk. Get Gly aligned along one side of the helix, and this is where the strands are in close proximity to each other. If Gly is replaced by even Ala, then too bulky and won t form the correct structure. The three chains can be held covalently held together by the formation of aldol cross links made by lysine oxidase. The amount of cross-linking increases with age leading to a loss of flexibility in joints. HyPro H-bonds across the triple helix. The hydroxylation of praline is a post-translational modification catalyzed by prolyl hydroxylase. This enzyme requires vitamin C as a cofactor. A dietary vitamin C deficiency leads to Scurvy which causes lesions in the skin and blood vessels because the structure of collagen is weakened. B) Lipoproteins Multicomponent complexes of proteins and lipids held together by non-covalent forces. An example is apolipoprotein A1, the main constituent in HDL and chylomicrons. This protein has a high a-helical content. The helical regions have amphipathic properties: every 3 rd or 4 th amino acid in the chain is charged and forms a polar edge that associates with the polar head groups of lipids and aqueous solvent. The opposite side of the helix has hydrophobic side chains which associate with the nonpolar tails of lipids (see figure 12.3). C) Glycoproteins These are proteins with sugar molecules covalently attached to the polypeptide structure. Most plasma proteins (except albumin) are glycoproteins. The carbohydrates can be distributed evenly along the peptide chain or concentrated in defined regions. Glycoproteins may have only one carbohydrate attached or multiple carbohydrates. Can attach chains of carbohydrates to the protein.

4 The addition of complex carbohydrate units occurs in a series of enzyme catalyzed reactions as the peptide chain is transported through the ER and Golgi network. Carbohydrate groups may be linked to polypeptide chains via: 1) the hydroxyl groups of Ser, Thr, hydroxylysine, or hydroxyproline (O-linked saccharides), 2)the amide group of Asn (N-linked saccharides), 3) the thiol side chain of Cys, or 4) the N-terminus of the peptide. Clinical: Form linkages between blood glucose and the N-terminus of hemoglobin in uncontrolled diabetes. Can assay for the concentration of glycosylated hemoglobin to follow changes in blood glucose concentration and follow the effectiveness of treatment. Protein Folding: Proteins fold into a single, unique 3-D structure based on the amino acid sequence and the noncovalent forces that act on the sequence. Folding is under thermodynamic and kinetic control. An exact knowledge of de novo folding of a polypeptide is at present unattainable; however, certain processes appear reasonable. unfolded protein small regions of secondary structure molten globular state native tertiary structure (short range noncovalent (a condensed intermed.) (lowest energy state) interactions) a-helix,β-structures rearrangements to reach lowest energy state form correct disulfide bonds Proteins that assist in the protein folding process: 1) cis-trans prolyl isomerase- catalyze interconversion between cis and trans peptide bonds of praline 2) protein disulfide isomerases- catalyze the breakage and formation of disulfide cystine linkages 3) Chaperone Proteins- heat shock proteins (synthesis increases at high temperatures) Do not change final outcome of folding process They do increase the rate of folding process by limiting the number of unproductive pathways available to a polypeptide. hsp 70- protect chain as it is being synthesized so that the polypeptide chain will not fold until the entire chain is synthesized. hsp 60- bind protein in the molten globular state within their central hydrophobic cavity to facilitate folding. Noncovalent Forces Noncovalent forces cause the peptide to fold into a unique conformation and then stabilize the native structure. These are weak bonding forces, but the large number of individually weak noncovalent contacts within a protein add up to a large energy factor that promotes protein folding. 1) Hydrophobic interaction forces- most important Nonpolar structures come together in aqueous solvent to minimize exposure to polar water. 2) Hydrogen bonds Hydrogen bonds increase the stability of a structure and lower its energy. Remember that the strength of the bond increases as the distance between decreases. Typically H-bond strengths in proteins are 1-7 kcal/mole. 3) Electrostatic interactions Get attractive and repulsive forces between charged groups. E el = Z a Z b ε 2 Z is the charge of each ion, D is the dielectric constant of the D r ab solvent, r ab is the distance between the charges, and ε is the charge of a proton or electron The dielectric constant of water is high (80); therefore, electrostatic interactions are weak in water but are high in the interior of the protein where D is low.

5 4) van der Waals- London dispersion and Dipole-Dipole These are the weakest individual forces, but there are so many interactions in the protein that these forces become very important. These forces are critical for secondary structure formation. Can have attractive and repulsive forces. The repulsive forces are the weakest when in a-helix or β-structure formation. Denaturation of Protein The loss of 2, 3, and 4 structure correlated with the loss of protein function. The loss of a single essential H-bond or electrostatic interaction or hydrophobic interaction can lead to the denaturation of the protein. change in ph, ionic strength of solvent, temperature, binding of prosthetic groups, cofactors, and substrate also affect protein stability substitution or modification of an essential amino acid that provides a critical noncovalent interaction dramatically affects the stability of a protein and can change the conformation of the protein. Can denature a protein in the lab with: DTT or β-mercaptoethanol-breaks cystine disulfide bonds Detergents-like SDS and guanidine- weaken hydrophobic interactions Strong base or acid changes the ionic strength of the solvent and affects electrostatic interactions Organic solvent Heating