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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.

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CHAPTER 5

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  1. 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

  2. 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

  3. 5.1 Proteins are Linear Polymers of Amino Acids

  4. 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

  5. The Coplanar Nature of the Peptide Bond Six atoms of the peptide group lie in a plane!

  6. “Peptides” • Short polymers of amino acids • Each unit is called a residue • 2 residues - dipeptide • 3 residues -tripeptide • 12-20 residues - oligopeptide • many - polypeptide

  7. “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

  8. 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!

  9. 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

  10. The sequence of ribonuclease A

  11. 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

  12. 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

  13. How to view a protein? • backbone only • backbone plus side chains • ribbon structure • space-filling structure

  14. Configuration and conformation are not the same

  15. 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

  16. The tetrameric structure of hemoglobin

  17. 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.

  18. 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

  19. 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.

  20. Determining the SequenceAn 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

  21. Determining the SequenceAn 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.

  22. Determining the SequenceAn Eight Step Strategy • 7. Reconstruct the sequence of the protein from the sequences of overlapping fragments • 8. Determine the positions of the disulfide crosslinks

  23. 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)

  24. 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

  25. 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)

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

  27. 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

  28. Steps 5 and 6: Fragmentation of the chains • Enzymatic fragmentation • trypsin, chymotrypsin, clostripain, staphylococcal protease • Chemical fragmentation • cyanogen bromide

  29. 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 thanLys • Staphylococcal protease • C-side of Glu, Asp in phosphate buffer • specific for Glu in acetate or bicarbonate buffer

  30. 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

  31. 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

  32. 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

  33. 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

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

  35. 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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