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

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 5.1 Proteins are Linear Polymers of Amino Acids

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 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Coplanar Nature of the Peptide Bond Six atoms of the peptide group lie in a plane!

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

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 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

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

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 sequence of ribonuclease A

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

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

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 Weak forces - H-bonds, ionic interactions, van der Waals interactions, hydrophobic interactions

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company How to view a protein? backbone only backbone plus side chains ribbon structure space-filling structure

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Configuration and conformation are not the same

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The tetrameric structure of hemoglobin

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

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

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

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

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

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Step 1: Separation of chains Subunit interactions depend on weak forces Separation is achieved with: - extreme pH - 8M urea - 6M guanidine HCl - high salt concentration (usually ammonium sulfate)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Step 2: Cleavage of Disulfide bridges Performic acid oxidation Sulfhydryl reducing agents - mercaptoethanol - dithiothreitol or dithioerythritol - to prevent recombination, follow with an alkylating agent like iodoacetate

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Step 3: Determine Amino Acid Composition described on pages 112,113 of G&G results often yield ideas for fragmentation of the polypeptide chains (Step 5, 6)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Step 4: Identify N- and C-terminal residues N-terminal analysis: –Edman's reagent –phenylisothiocyanate –derivatives are phenylthiohydantions –or PTH derivatives

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Step 4: Identify N- and C-terminal residues C-terminal analysis – Enzymatic analysis (carboxypeptidase) –Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys –Carboxypeptidase B (hog pancreas) only works on Arg and Lys

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Steps 5 and 6: Fragmentation of the chains Enzymatic fragmentation –trypsin, chymotrypsin, clostripain, staphylococcal protease Chemical fragmentation –cyanogen bromide

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Enzymatic Fragmentation Trypsin - cleavage on the C-side of Lys, Arg Chymotrypsin - C-side of Phe, Tyr, Trp Clostripain - like trypsin, but attacks Arg more than Lys Staphylococcal protease –C-side of Glu, Asp in phosphate buffer –specific for Glu in acetate or bicarbonate buffer

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chemical Fragmentation Cyanogen bromide CNBr acts only on methionine residues CNBr is useful because proteins usually have only a few Met residues see Fig for mechanism be able to recognize the results! –a peptide with a C-terminal homoserine lactone

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 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Step 7: Reconstructing the Sequence Use two or more fragmentation agents in separate fragmentation experiments Sequence all the peptides produced (usually by Edman degradation) Compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chain

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Reconstructing the Sequence Compare cleavage by trypsin and staphylococcal protease on a typical peptide: Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K Staphylococcal protease: F-S-G-I-T-P-K L-V-G-K-A-E

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Reconstructing the Sequence The correct overlap of fragments: L-V-G-K A-E-F-S-G-I-T-P-K L-V-G-K-A-E F-S-G-I-T-P-K Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Sequence analysis of catrocollastatin-C, a 23.6 kD protein from the venom of Crotalus atrox

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

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 more hydrophobic residues, whereas fibrous proteins may have atypical sequences Homologous proteins from different organisms have homologous sequences e.g., cytochrome c is highly conserved

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 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

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 Copyright © 1999 by Harcourt Brace & Company Laboratory Synthesis of Peptides Strategies are complex because of the need to control side chain reactions Blocking groups must be added and later removed du Vigneaud’s synthesis of oxytocin in 1953 was a milestone Bruce Merrifield’s solid phase method was even more significant

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Solid Phase Synthesis Carboxy terminus of a nascent peptide is covalently anchored to an insoluble resin After each addition of a residue, the resin particles are collected by filtration Automation and computer control now permit synthesis of peptides of 30 residues or more

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