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Protein Geometry
CORN LAW amino acid with L configuration
Greek alphabet
Chapter 5
6. Cellular defense immuoglobulins Antibodies Killer T cell Receptors 7. Structural Collagen Silk, etc. Function is dictated by protein structure!!
1 = Amino acid sequence, the linear order of AAs. Remember from the N-terminus to the C-terminus Above all else this dictates the structure and function of the protein.
3. Tertiary Structure 3 = the 3 dimensional structure of an entire peptide. Great in detail but vague to generalize. Can reveal the detailed chemical mechanisms of an enzyme.
4. Quaternary Structure 4 two or more peptide chains associated with a protein. Spatial arrangements of subunits. Chapter 5.3 is how to determine a proteins primary structure.
Protein Chemistry
It took 10 years, many people, and it took 100 g of protein! Today it takes one person several days to sequence the same insulin. 1021 AA b- glactosidase 1978
1. Prepare protein for sequencing a. Determine number of chemically different polypeptides. b. Cleave the proteins disulfide bonds. c. Separate and purify each subunit. d. Determine amino acid composition for each peptide.
At best, the automated instruments can sequence about 50 amino acids in one run!
Proteins must be cleaved into smaller pieces to obtain a complete sequence.
Bovine insulin should give 2 N-terminii and 2 Cterminii N-terminus 1-Dimethylamino - naphthalene-5-sulfonyl chloride Dansyl chloride Reacts with amines: N-terminus + Lys (K) side chains
Disadvantage with the Dansyl-chloride method is that you must use 6M HCl to cleave off the derivatized amino acid, this also cleaves all other amide bonds (residues) as well.
Edman degradation has been automated as a method to sequence proteins. The PTH-amino acid is soluble in solvents that the protein is not. This fact is used to separate the tagged amino acid from the remaining protein, allowing the cycle of labeling, degradation, and separation to continue.
Even with the best chemistry, the reaction is about 98% efficient. After sufficient cycles more than one amino acid is identified, making the sequence determination error-prone at longer reads.
Carboxypeptidase
Rn-2 O NH CH C NH
Rn-1 CH
O C O H3 N
Rn CH
O C
Carboxypeptidase A Rn-1 P
Rn R, K, P
If the Tyr-Ser bond is more resistant to cleavage than the Leu-Tyr, the Ser and the Tyr will appear simultaneously and the C-terminus would still be in doubt.
-S-S-
2SH
Also
Gln and Asn yield Glu and Asp
Base hydrolysis 2 to 4 N NaOH at 100 oC for 4 - 8 h. Is problematic, destroys Cys Ser, Thr, Arg but does not harm Trp.
H3N
CH
CH2
CH2
R
OH
O C O
CH
Scissile Bond
Trypsin
Endopeptidase V8
Cleave the large protein using i.e trypsin, separate fragments and sequence all of them. (We do not know the order of the fragments!!) Cleave with a different reagent i.e. Cyanogen Bromide, separate the fragments and sequence all of them. Align the fragments with overlapping sequence to get the overall sequence.
H3N-
_-_-_-_-_-_-_-_-_-_-_-_-_-_-COO
10
11
12
13
14
K F-A-M-K K-F-A-M Q-M-K D-I-K-Q-M G-M-D-I-K Y-R-G-M Y-R Trypsin cleaves after K or R Cyanogen Bromide (CN (positively charged amino Br) Cleaves after Met acids) i.e M - X Q-M-K D-I-K-Q-M G-M-D-I-K K F-A-M-K K-F-A-M Y-R Y-R-G-M
Size
Charge
Solubility
Chemical specificity Hydrophobicity/ Hydrophylicity Reverse Phase High Pressure Liquid Chromatography is used to separate peptide fragments.
Peptide mapping: digest protein with an appropriate agent, then separate using two dimensional paper chromatography
Digested Peptide from normal (HbA) and Sickle cell anemia (Hbs) hemoglobins
HbA
HbS
V-H-L-T-P-E-E-K
V-H-L-T-P-V-E-K
1 2 3 4 5 6 7 8
Deoxyhemoglobin aggregates and deforms cell. Primary structure changes dictate quaternary structure. Why did the problem not die out? Homozygotic normal gets malaria Heterzyatic sickle cell trait resistant to malaria Homozygotic sickle cell gets sickle cell
dies
dies
Homologous proteins
(evolutionarily related proteins)
Compare protein sequences: Conserved residues, i.e invariant residues reflect chemical necessities. Conserved substitutions, substitutions with similar chemical properties Asp for Glu, Lys for Arg, Ile for Val
0 2 6 9 9 7 19 8 11 17 22 27 44 46 45 50
5.1 0 6 8 8 6 18 9 11 17 21 26 44 46 45 50 0 12 10 10 21 11 13 18 24 28 47 49 46 51
0 2 3 19 8 11 17 23 28 46 47 46 51
0 3 20 8 12 18 24 27 46 48 45 50
0 17 7 11 17 22 27 46 46 46 51
Phylogenetic tree
Indicates the ancestral relationships among the organisms that produced the protein. Each branch point indicates a common ancestor. Relative evolutionary distances between neighboring branch points are expressed as the number of amino acid differences per 100 residues of the protein. PAM units or Percentage of Accepted Mutations
Although DNA mutates at an assumed constant rate. Some proteins cannot accept mutations because the mutations kill the function of the protein and thus are not viable.
Although insects have shorter generation times than mammals and many more rounds of replication, the number of mutations appear to be independent of the number of generations but dependent upon time
Cytochrome c amino acid differences between mammals, insects and plants note the similar distances
Hemoglobin:
is an oxygen transport protein it must bind and release oxygen as the cells require oxygen
Myoglobin:
is an oxygen storage protein
it binds oxygen tightly and releases it when oxygen concentrations are very low
3. Developed a tetrameric structure two a and two b chains increased oxygen transport capabilities.
4. Mammals have fetal hemoglobin with a variant b chain i.e. g (a2g2). 5. Human embryos contain another hemoglobin 2e2. 6. Primates also have a d chain with no known unique function.
Protein Evolution is not organismal evolution Chimpanzee human are about 99% the same amino acid sequences in proteins! However: Rapid divergence with few mutational changes suggest altered control of gene expression.