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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
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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

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Biochemistry 2/e - Garrett & Grisham 5.1 Proteins are Linear Polymers of Amino Acids

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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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

The Coplanar Nature of the Peptide Bond

Six atoms of the peptide group lie in a plane!

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Biochemistry 2/e - Garrett & Grisham

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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

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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

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Biochemistry 2/e - Garrett & Grisham

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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!
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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
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Biochemistry 2/e - Garrett & Grisham

The sequence of ribonuclease A

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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

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Biochemistry 2/e - Garrett & Grisham

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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

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

How to view a protein?


backbone only backbone plus side chains ribbon structure space-filling structure

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham Configuration and conformation are not the same

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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

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Biochemistry 2/e - Garrett & Grisham

The tetrameric structure of hemoglobin

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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.
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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
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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.

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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
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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.

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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

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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)
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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

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)

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Biochemistry 2/e - Garrett & Grisham

Step 4:
Identify N- and C-terminal residues N-terminal analysis:
Edman's reagent phenylisothiocyanate derivatives are phenylthiohydantions or PTH derivatives

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

Steps 5 and 6:
Fragmentation of the chains Enzymatic fragmentation
trypsin, chymotrypsin, clostripain, staphylococcal protease

Chemical fragmentation
cyanogen bromide

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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

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

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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Biochemistry 2/e - Garrett & Grisham

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 Vigneauds synthesis of oxytocin in 1953 was a milestone Bruce Merrifields solid phase method was even more significant
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Biochemistry 2/e - Garrett & Grisham

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
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Biochemistry 2/e - Garrett & Grisham

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