Biochemistry 2 e Garrett Grisham CHAPTER 5 Proteins Their Biological Functions and Primary Structure to accompany Biochemistry 2 e by Reginald Garrett and Charles Grisham All rights reserved Requests for permission to make copies of any part of the work should be mailed to Permissions Department Harcourt Brace Company 6277 Sea Harbor Drive Orlando Florida 32887 6777 Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham Outline 5 1 Proteins Linear Polymers of Amino Acids 5 2 Architecture 5 3 Many Biological Functions 5 4 May be Conjugated with Other Groups 5 7 Primary Structure Determination 5 8 Consider the Nature of Sequences Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham 5 1 Proteins are Linear Polymers of Amino Acids 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 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 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 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 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 sequence of ribonuclease A Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham 5 2 Architecture of Proteins Shape globular or fibrous The levels of protein structure Primary sequence Secondary local structures H bonds Tertiary overall 3 dimensional shape Quaternary subunit organization Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham 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 Weak forces H bonds ionic interactions van der Waals interactions hydrophobic interactions Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham How to view a protein backbone only backbone plus side chains ribbon structure space filling structure Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham 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 5 3 Biological Functions of Proteins Proteins are the agents of biological function Enzymes Ribonuclease Regulatory proteins Insulin Transport proteins Hemoglobin Structural proteins Collagen Contractile proteins Actin Myosin Exotic proteins Antifreeze proteins in fish Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham The tetrameric structure of hemoglobin Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham 5 4 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 5 7 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 Insulin consists of two polypeptide chains A and B held together by two disulfide bonds The A chain has 21 residues and the B chain has 30 residues The sequence shown is that of bovine insulin Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham Determining the Sequence An Eight Step Strategy 1 If more than one polypeptide chain separate 2 Cleave reduce disulfide bridges 3 Determine composition of each chain 4 Determine N and C terminal residues Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham Determining the Sequence An Eight Step Strategy 5 Cleave each chain into smaller fragments and determine the sequence of each chain 6 Repeat step 5 using a different cleavage procedure to generate a different set of fragments Copyright 1999 by Harcourt Brace Company Biochemistry 2 e Garrett Grisham Determining the Sequence An Eight Step Strategy 7 Reconstruct the sequence of the protein from the sequences of overlapping fragments 8 Determine the positions of the disulfide crosslinks Copyright 1999 by Harcourt Brace Company