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Biochemistry 2/e - Garrett & Grisham Proteins: Their Structure and Biological Functions Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Biological Functions of Proteins • • • • • • Proteins are the agents of biological function Enzymes - Ribonuclease Regulatory proteins - Insulin, PCNA Transport proteins - Hemoglobin Structural proteins - Collagen Contractile proteins - Actin, Myosin Protective proteins - Antifreeze proteins Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Protein structure often provides clues about protein function Unrelated proteins assume similar structures to fulfill common functions Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Proteins are Linear Polymers of Amino Acids Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Peptides • • • • • • Short polymers of amino acids Each unit is called a residue 2 residues - dipeptide 3 residues - tripeptide 12-20 residues - oligopeptide many - polypeptide Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Protein One or more polypeptide chains • One polypeptide chain - a monomeric protein • • • • More than one - multimeric protein Homomultimer - one kind of chain Heteromultimer - two or more different chains Hemoglobin, for example, is a heterotetramer; it has two alpha chains and two beta chains Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Proteins - Large and Small • Insulin - A chain of 21 residues, B chain of 30 residues -total mol. wt. of 5,733 • Glutamine synthetase - 12 subunits of 468 residues each - total mol. wt. of 600,000 • Connectin proteins - alpha - MW 2.8 million! • beta connectin - MW of 2.1 million, with a length of 1000 nm -it can stretch to 3000 nm! Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Amino acid composition provides some (limited) clues about protein structure-function Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham The Sequence of Amino Acids in a Protein • is a unique characteristic of every protein • is encoded by the nucleotide sequence of DNA • is thus a form of genetic information • is read from the amino terminus to the carboxyl terminus Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham • The levels of protein structure - Primary sequence - Secondary local structures - Tertiary overall 3-dimensional shape - Quaternary subunit organization Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham What forces determine the structure? • Primary structure - determined by covalent bonds • Secondary, Tertiary, Quaternary structures - all determined by weak forces Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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. Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham The sequence of ribonuclease A Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Sequence Determination Frederick Sanger was the first - in 1953, he sequenced the two chains of insulin. • Sanger's results established that all of the molecules of a given protein have the same sequence • Proteins can be sequenced in two ways: - real amino acid sequencing - sequencing the corresponding DNA in the gene Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Nature of Protein Sequences • Sequences and composition reflect the function of the protein: • Membrane proteins have stretches of hydrophobic residues, whereas fibrous proteins may have atypical sequences • Homologous proteins from different organisms have similar sequences e.g., cytochrome c is highly conserved Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Phylogeny of Cytochrome c • The number of amino acid differences between two cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derived • This observation can be used to build phylogenetic trees of proteins • This is the basis for studies of molecular evolution Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham So, how do proteins fold? Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Proteins are Linear Polymers of Amino Acids Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham The Coplanar Nature of the Peptide Bond Six atoms of the peptide group lie in a plane Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Configuration and conformation are not the same Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Secondary Structure 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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. Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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) Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham The Alpha Helix • First proposed by Linus Pauling and Robert Corey in 1951 • A ubiquitous component of proteins • Stabilized by H-bonds Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham The Beta-Pleated Sheet Composed of beta strands • Also first postulated by Pauling and Corey, 1951 • Strands may be parallel or antiparallel Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Steric Constraints on phi & psi • G. N. Ramachandran was the first to demonstrate the convenience of plotting phi,psi combinations from known protein structures • The sterically favorable combinations are the basis for preferred secondary structures Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Predictive Algorithms If the sequence holds the secrets of folding, can we figure it out? Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Tertiary Structure So, how do proteins fold? Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Weak Forces are Responsible for Protein Folding • • • • 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Thermodynamics of Folding • Separate the enthalpy and entropy terms for the peptide chain and the solvent Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham The largest favorable contribution to folding is the entropy term for the interaction of nonpolar residues with the solvent Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 • Chaperones may help • Peptide chains, composed of L-amino acids, have a tendency to undergo a "right-handed twist" Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Globular Proteins • • • • More design principles "Random coil" is not random Structures of globular proteins are not static Various elements of protein move to different degrees Some segments of proteins are very flexible and disordered Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham An amphiphilic helix in flavodoxin: A nonpolar helix in citrate synthase: A polar helix in calmodulin: Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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) Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Other Chemical Groups in Proteins Proteins may be "conjugated" with other chemical groups • If the non-amino acid part of the protein is important to its function, it is called a prosthetic group. • Be familiar with the terms: glycoprotein, lipoprotein, nucleoprotein, phosphoprotein, metalloprotein, hemoprotein, flavoprotein. Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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! Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company