Chapter 5 Proteins: Their Biological Functions and Primary Structure

CHAPTER 5

• Proteins: Their Biological Functions and Primary Structure

• to accompany

• Biochemistry, 2/e

• by

• Reginald Garrett and Charles 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

5.1 Proteins are Linear Polymers of Amino Acids

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

The Coplanar Nature of the Peptide Bond

• Six atoms of the peptide group lie in a plane!

“Peptides”

• Short polymers of amino acids

• Each unit is called a residue

• 2 residues - dipeptide

• 3 residues - tripeptide

• 12-20 residues - oligopeptide

• many - polypeptide

“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

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!

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

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

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

How to view a protein?

• backbone only

• backbone plus side chains

• ribbon structure

• space-filling structure

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

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.

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

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

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.

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

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)

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

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)

Step 4:

• Identify N- and C-terminal residues

• N-terminal analysis:

• Edman's reagent
• phenylisothiocyanate
• derivatives are phenylthiohydantions
• or PTH derivatives

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

Steps 5 and 6:

• Fragmentation of the chains

• Enzymatic fragmentation

• trypsin, chymotrypsin, clostripain, staphylococcal protease

• Chemical fragmentation

• cyanogen bromide

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

Chemical Fragmentation

• Cyanogen bromide

• CNBr acts only on methionine residues

• CNBr is useful because proteins usually have only a few Met residues

• see Fig. 5.21 for mechanism

• be able to recognize the results!

• a peptide with a C-terminal homoserine lactone

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

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

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

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

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

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

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