Lecture3&4.ppt

The Role of the Sequence in Protein Structure All of the information necessary for folding the peptide chain into its "native? structure is contained in the primary amino acid structure of the peptide. The sequence of ribonuclease A Pure proteins can be crystallized, therefore they adopt the same structure Primary structure - determined by covalent bonds Secondary, Tertiary, Quaternary structures - all determined by weak forces What forces determine the structure? Outline Forces Influencing Protein Structure Role of the Amino Acid Sequence in Protein Structure Secondary Structure of Proteins Protein Folding and Tertiary Structure Subunit Interactions and Quaternary Structure The Weak Forces What are they? What are the relevant numbers? van der Waals: 0.4 - 4 kJ/mol hydrogen bonds: 12-30 kJ/mol ionic bonds: 20 kJ/mol hydrophobic interactions: <40 kJ/mol Proteins are Linear Polymers of Amino Acids The atoms of the peptide bond lie in a plane The resonance stabilization energy of the planar structure is 88 kJ/mol A twist about the C-N bond involves a twist energy of 88 kJ/mol times the square of the twist angle. Twists can occur about either of the bonds linking the alpha carbon to the other atoms of the peptide backbone Six atoms of the peptide group lie in a plane The Coplanar Nature of the Peptide Bond Most peptide bonds are in the trans configuration Configuration and conformation are not the same The Peptide Bond is usually found in the trans conformation has partial (40%) double bond character is about 0.133 nm long - shorter than a typical single bond but longer than a double bond Due to the double bond character, the six atoms of the peptide bond group are always planar. N partially positive; O partially negative Consequences of the Amide Plane Two degrees of freedom per residue for the peptide chain Angle about the C(alpha)-N bond is denoted phi Angle about the C(alpha)-C bond is denoted psi The entire path of the peptide backbone is known if all phi and psi angles are specified Some values of phi and psi are more likely than others. Steric Constraints on phi & psi Unfavorable overlap precludes some combinations of phi and psi phi = 0, psi = 180 is unfavorable phi = 180, psi = 0 is unfavorable phi = 0, psi = 0 is unfavorable Steric Constraints on phi & psi G. N. Ramachandran was the first to demonstrate the convenience of plotting phi,psi combinations from known protein structures Ramachandran plot for L-Ala residues The sterically favorable combinations of phi and psi are the basis for preferred secondary structures Classes of Secondary Structure All these are local structures that are stabilized by hydrogen bonds Alpha helix Beta sheet (composed of "beta strands") Tight turns (aka beta turns or beta bends) The Alpha Helix First proposed by Linus Pauling and Robert Corey in 1951 Identified in keratin by Max Perutz A ubiquitous component of proteins Stabilized by H-bonds The Alpha Helix Residues per turn: 3.6 Rise per residue: 1.5 Angstroms Rise per turn (pitch): 3.6 x 1.5A = 5.4 Angstroms The backbone loop that is closed by any H-bond in an alpha helix contains 13 atoms phi = -60 degrees, psi = -45 degrees The non-integral number of residues per turn Helical handedness The Beta-Pleated Sheet Composed of beta strands Also first postulated by Pauling and Corey, 1951 Strands may be parallel or antiparallel Rise per residue: 3.47 Angstroms for antiparallel strands 3.25 Angstroms for parallel strands Each strand of a beta sheet may be pictured as a helix with two residues per turn The Beta Turn (aka beta bend, tight turn) allows the peptide chain to reverse direction carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away proline and glycine are prevalent in beta turns Secondary structure predictions So, how do proteins fold? Tertiary Structure Tertiary Structure Several important principles: Secondary structures form wherever possible (due to formation of large numbers of H-bonds) Helices and sheets often pack close together A hydrophobic core is formed Tertiary Structure Several important principles: The backbone links between elements of secondary structure are usually short and direct Proteins fold to make the most stable structures (make H-bonds and minimize solvent contact Stable folding patterns Peptide chains, composed of L-amino acids, have a tendency to undergo a "right-handed twist" Stable folding patterns Stable folding patterns Stable folding patterns Antiparallel Alpha Helical Proteins Simplest way to pack helices - short connecting loops and antiparallel packing The helix bundle often involves a slight (15 degree) left-handed twist The globin proteins - myoglobin and hemoglobin - are antiparallel alpha proteins An amphiphilic helix in flavodoxin: A nonpolar helix in citrate synthase: A polar helix in calmodulin: How do proteins recognize and interpret the folding information? Certain loci along the chain may act as nucleation points Protein chain must avoid local energy minima Thermodynamics of Folding Separate the enthalpy and entropy terms for the peptide chain and the solvent Further distinguish polar and nonpolar groups The largest favorable contribution to folding is the entropy term for the interaction of nonpolar residues with the solvent Molecular Chaperones Why are chaperones needed if the information for folding is inherent in the sequence? to protect nascent proteins from the concentrated protein matrix in the cell and perhaps to accelerate slow steps Chaperone proteins were first identified as "heat-shock proteins" (hsp60 and hsp70) Protein Modules An important insight into protein structure Many proteins are constructed as a composite of two or more "modules" or domains Each of these is a recognizable domain that can also be found in other proteins Sometimes modules are used repeatedly in the same protein There is a genetic basis for the use of modules in nature Unrelated proteins assume similar structures to fulfill common functions Protein structure often provides clues about protein function Fibrous Proteins Much or most of the polypeptide chain is organized approximately parallel to a single axis Fibrous proteins are often mechanically strong Fibrous proteins are usually insoluble Usually play a structural role in nature Globular Proteins Some design principles Most polar residues face the outside of the protein and interact with solvent Most hydrophobic residues face the interior of the protein and interact with each other Packing of residues is close However, ratio of vdw volume to total volume is only 0.72 to 0.77, so empty space exists The empty space is in the form of small cavities Globular Proteins More design principles "Random coil" is not random Structures of globular proteins are not static Various elements and domains of protein move to different degrees Some segments of proteins are very flexible and disordered Know the kinds and rates of protein motion Globular Proteins The Forces That Drive Folding Peptide chain must satisfy the constraints inherent in its own structure Peptide chain must fold so as to "bury" the hydrophobic side chains, minimizing their contact with water Quaternary Structure What are the forces driving quaternary association? Typical Kd for two subunits: 10-8 to 10-16M! These values correspond to energies of 50-100 kJ/mol at 37 C Entropy loss due to association - unfavorable Entropy gain due to burying of hydrophobic groups - very favorable! What are the structural and functional advantages driving quaternary association? Know these! Stability: reduction of surface to volume ratio Genetic economy and efficiency Bringing catalytic sites together Cooperativity Predictive Algorithms If the sequence holds the secrets of folding, can we figure it out? Many protein chemists have tried to predict structure based on sequence Chou-Fasman: each amino acid is assigned a "propensity" for forming helices or sheets Chou-Fasman is only modestly successful and doesn't predict how sheets and helices arrange George Rose may be much closer to solving the problem. See Proteins 22, 81-99 (1995)