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, Sea Harbor Drive, Orlando, Florida

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 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

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

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

9. One or more polypeptide chains
“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

11. 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!

13. 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

14. The sequence of ribonuclease A

15. 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

17. 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

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

21. Configuration and conformation are not the same

22. 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

23. The tetrameric structure of hemoglobin

24. 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.

25. 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

26. 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.

27. 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

28. 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.

29. 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

30. 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)

31. Cleavage of Disulfide bridges
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

33. Determine Amino Acid Composition
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)

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

36. Identify N- and C-terminal residues
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

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

38. 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

40. Chemical Fragmentation
Cyanogen bromide
CNBr acts only on methionine residues
CNBr is useful because proteins usually have only a few Met residues
see Fig for mechanism
be able to recognize the results!
a peptide with a C-terminal homoserine lactone

45. Reconstructing the Sequence
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

46. 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

47. 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

48. Sequence analysis of catrocollastatin-C, a 23
Sequence analysis of catrocollastatin-C, a 23.6 kD protein from the venom of Crotalus atrox

50. 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

53. 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

58. 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

59. 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