Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Proteins: Their Structure and Biological Functions.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Unrelated proteins assume similar structures to fulfill common functions Protein structure often provides clues about protein function

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Peptides Short polymers of amino acids Each unit is called a residue 2 residues - dipeptide 3 residues - tripeptide 12-20 residues - oligopeptide many - polypeptide

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The levels of protein structure - Primary sequence - Secondary local structures - Tertiary overall 3-dimensional shape - Quaternary subunit organization

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company What forces determine the structure? Primary structure - determined by covalent bonds Secondary, Tertiary, Quaternary structures - all determined by weak forces

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Alpha Helix First proposed by Linus Pauling and Robert Corey in 1951 A ubiquitous component of proteins Stabilized by H-bonds

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Beta-Pleated Sheet Composed of beta strands Also first postulated by Pauling and Corey, 1951 Strands may be parallel or antiparallel

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The largest favorable contribution to folding is the entropy term for the interaction of nonpolar residues with the solvent

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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"

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Quaternary Structure What are the forces driving quaternary association? Typical K d for two subunits: 10 -8 to 10 -16 M! 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!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Proteins: Their Structure and Biological Functions.

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Proteins: Their Structure and Biological Functions.

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