Chapter 5 Proteins: Their Biological Functions and Primary Structure


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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!











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






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