CHAPTER 5Proteins: Their BiologicalFunctions and Primary

Structure

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5.1 Proteins are Linear Polymers of Amino AcidsProteins are linear polymers of amino acids:

The Peptide BondThe Peptide Bond• Bond occurs between the αα-amino group of one amino acid and the αα-carboxyl group of another amino acid

• A condensation reaction where the elements of H20 are removed

N N - C - COOH

H

H

HH

HHC - OHNH2 - C - C - OH

H

H OO

N N - C - COOH

H

H

HH

C NH2 - C - C

H

H OO

H H -OH- OH

C NH2 - C - C

H

H OO

N N - C - COOH

H

H

HH

The Peptide Bond!!The Peptide Bond!!

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 typicalsingle bond but longer than a double bond

• Due to the double bond character, the sixatoms of the peptide bond group are alwaysplanar!

• N partially positive; O partially negative

THE PEPTIDE BOND:

C NH2 - C - C

H

H OO

N N - C - COOH

H

H

HH

The Peptide Bond is a Resonance Structure:

C NH2 - C - C

H

H O-O-

N+ N+ - C - COOH

H

H

HH

The Coplanar Nature of the Peptide Bond

Six atoms of the peptide group lie in a plane!

The coplanar nature of the Peptide Bond

“Peptides”

• Short polymers of amino acids

• Each unit is called a residue

• 2 residues - dipeptide

• 3 residues - tripeptide

• 12-20 residues - oligopeptide

• many - polypeptide

“Peptides”

“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

“Protein”

The tetrameric structure of hemoglobin

Proteins - Large and Small

• Insulin - A chain of 21 residues, B chain of 30residues -total mol. wt. of 5,733

• Glutamine synthetase - 12 subunits of 468residues each - total mol. wt. of 600,000

• Connectin proteins - alpha - MW 2.8 million!

• beta connectin - MW of 2.1 million, with alength of 1000 nm -it can stretch to 3000 nm!

Proteins - Large and Small

The Sequence of Amino Acids ina Protein

• is a unique characteristic of every protein

• is encoded by the nucleotide sequence ofDNA

• is thus a form of genetic information

• is read from the amino terminus to thecarboxyl terminus

The Sequence of Amino Acidsin a Protein:

• Protein chains have a direction.• Protein chains have a direction.

• By convention the N-terminus is taken to be the beginning of a polypeptide chain.

• By convention the N-terminus is taken to be the beginning of a polypeptide chain.

NH2 - C - C - N - C - C -N - C - COOHNH

2 - C - C - N - C - C -N - C - COOH

OO OOHH

HHHH HH

HH HH

HH CH3

CH3

Glycine-Glycine-AlanineGlycine-Glycine-Alanine

The sequence of ribonuclease A

5.2 Architecture of Proteins

• Shape - globular, fibrous, membrane

• The levels of protein structure

- Primary - linear amino acid sequence

- Secondary - peptide backbone - H-bonds

- Tertiary - overall 3-dimensional shape

- Quaternary - subunit organization

Architecture of Proteins

What forces determine thestructure?

• Primary structure - determined by covalent bonds

• Secondary, Tertiary, Quaternary structures - alldetermined by weak forces

• Weak forces - H-bonds, ionic interactions, vander Waals interactions, hydrophobic interactions

What forces determine the structure?

The tetrameric structure of hemoglobin

How to view a protein?

• backbone only

• backbone plus side chains

• ribbon structure

• space-filling structure

How to View a Protein?

Configuration andconformation arenot the same

5.3 Biological Functions ofProteinsProteins 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

• Storage - seed storage proteins in plants

Biological Functions of Proteins:

5.4 Other Chemical Groups inProteins

Proteins may be "conjugated" with otherchemical groups

• If the non-amino acid part of the protein isimportant to its function, it is called aprosthetic group.

• Large organic molecules (vitamins)comjugated to proteins are coenzymes.

• Be familiar with the terms: glycoprotein,lipoprotein, nucleoprotein, phosphoprotein,metalloprotein, hemoprotein, flavoprotein.

Other Chemical Groups in Proteins:

5.7 Sequence DeterminationFrederick Sanger was the first - in 1953, he

sequenced the two chains of insulin.

• Sanger's results established that all of themolecules of a given protein have the samesequence.

• Proteins can be sequenced in two ways:

- real amino acid sequencing

- sequencing the corresponding DNA in the gene

Sequence Determination

Insulin consists of twopolypeptide chains, Aand B, held together bytwo disulfide bonds.The A chain has 21residues and the Bchain has 30 residues.

The sequence shown isthat of bovine insulin.

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:

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:

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

Determining the Sequence:

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 1:

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 2:

Step 3:

Determine Amino Acid Composition

• described on pages 112,113 of G&G

• results often yield ideas for fragmentation ofthe polypeptide chains (Step 5, 6)

Step 3:

Step 4:

Identify N- and C-terminal residues

• N-terminal analysis:– Edman's reagent

– phenylisothiocyanate

– derivatives are phenylthiohydantions

– or PTH derivatives

Step 4:

Step 4:

Identify N- and C-terminal residues

• C-terminal analysis– Enzymatic analysis (carboxypeptidase)

– Carboxypeptidase A cleaves any residue exceptPro, Arg, and Lys

– Carboxypeptidase B (hog pancreas) only works onArg and Lys

Step 4:

Steps 5 and 6:

Fragmentation of the chains

• Enzymatic fragmentation– trypsin, chymotrypsin, clostripain,

staphylococcal protease

• Chemical fragmentation– cyanogen bromide

Steps 5 & 6:

Enzymatic Fragmentation

• Trypsin - cleavage on the C-side of Lys, Arg

• Chymotrypsin - C-side of Phe, Tyr, Trp

• Clostripain - like trypsin, but attacks Arg morethan Lys

• Staphylococcal protease– C-side of Glu, Asp in phosphate buffer

– specific for Glu in acetate or bicarbonate buffer

Enzymatic Fragmentation

Chemical Fragmentation

Cyanogen bromide

• CNBr acts only on methionine residues

• CNBr is useful because proteins usuallyhave 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

Chemical Fragmentation

Step 7:

Reconstructing the Sequence

• Use two or more fragmentation agents inseparate fragmentation experiments

• Sequence all the peptides produced (usuallyby Edman degradation)

• Compare and align overlapping peptidesequences to learn the sequence of theoriginal polypeptide chain

Step 7:

Reconstructing the Sequence

Compare cleavage by trypsin andstaphylococcal 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

Reconstructing the Sequence

• The correct overlap of fragments:

L-V-G-K A-E-F-S-G-I-T-P-KL-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

Reconstructing the Sequence

Sequence analysis of catrocollastatin-C, a 23.6 kDprotein from the venom of Crotalus atrox

Nature of Protein Sequences

• Sequences and composition reflect thefunction of the protein

• Membrane proteins have more hydrophobicresidues, whereas fibrous proteins may haveatypical sequences

• Homologous proteins from differentorganisms have homologous sequences

• e.g., cytochrome c is highly conserved

Nature of Protein Seqences

Phylogeny of Cytochrome c

• The number of amino acid differencesbetween two cytochrome c sequences isproportional to the phylogenetic differencebetween the species from which they arederived

• This observation can be used to buildphylogenetic trees of proteins

• This is the basis for studies of molecularevolution

Phylogeny of Cytochrome C

Laboratory Synthesis of Peptides

• Strategies are complex because of the needto control side chain reactions

• Blocking groups must be added and laterremoved

• du Vigneaud’s synthesis of oxytocin in1953 was a milestone

• Bruce Merrifield’s solid phase method waseven more significant

Laboratory Synthesis of Peptides

Solid Phase Synthesis

• Carboxy terminus of a nascent peptide iscovalently anchored to an insoluble resin

• After each addition of a residue, the resinparticles are collected by filtration

• Automation and computer control nowpermit synthesis of peptides of 30 residuesor more

Laboratory Synthesis of Peptides