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CHAPTER 5. Proteins: Their Biological Functions and Primary Structure to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham.

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Chapter 5

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


Outline
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



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 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
The Coplanar Nature of the Peptide Bond

Six atoms of the peptide group lie in a plane!


Peptides
“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
“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
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
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
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
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
How to view a protein?

  • backbone only

  • backbone plus side chains

  • ribbon structure

  • space-filling structure



5 3 biological functions of proteins
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
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
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


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.


Determining the sequence an eight step strategy
Determining the Sequence together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.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 strategy1
Determining the Sequence together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.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 strategy2
Determining the Sequence together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.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
Step 1: together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

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
Step 2: together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

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
Step 3: together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

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
Step 4: together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

Identify N- and C-terminal residues

  • N-terminal analysis:

    • Edman's reagent

    • phenylisothiocyanate

    • derivatives are phenylthiohydantions

    • or PTH derivatives


Step 41
Step 4: together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

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
Steps 5 and 6: together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

Fragmentation of the chains

  • Enzymatic fragmentation

    • trypsin, chymotrypsin, clostripain, staphylococcal protease

  • Chemical fragmentation

    • cyanogen bromide


Enzymatic fragmentation
Enzymatic Fragmentation together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

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

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

  • Clostripain - like trypsin, but attacks Arg more thanLys

  • Staphylococcal protease

    • C-side of Glu, Asp in phosphate buffer

    • specific for Glu in acetate or bicarbonate buffer


Chemical fragmentation
Chemical Fragmentation together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

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
Step 7: together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

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
Reconstructing the Sequence together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

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 sequence1
Reconstructing the Sequence together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.

  • 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
Nature of Protein Sequences from the venom of

  • 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
Phylogeny of Cytochrome c from the venom of

  • 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
Laboratory Synthesis of Peptides from the venom of

  • 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
Solid Phase Synthesis from the venom of

  • 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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