1. Chap. 5 AA & Scoring Matrix

2. DNA Bases s R Y W M K B N H V D A T G C A T G C A T G C A T G C A T

3. A G T C Models of nucleotide substitution transition Purine =>
transversion
transversion
Pyrimidine => T C
transition

4. Jukes-Cantor (JC) Kimura 2P Kimura

5. Point Mutation (Substitution)
Point mutation – simplest form of mutation and occurs all over DNA sequences
Transition – mutation within purine (A,G) or pyrimidine (C,T/U)
Transversion – mutation between nt groups
Effects depend on where mutations occur
Non-coding region – no effect on proteins, and neutral
But may have significant effects if occurring in control region
Coding region
Synonymous substitution when a mutation does not change AA
Non-synonymous
AA is replaced by another
stop codon is introduced

6. Other Mutations Indel mutation
Small indels of a single base of a few bases are frequent
Particularly frequent with repeated sequences
GCGC…: insertion of extra GC or deletion cause slight slippage
CAG repeated region in huntingtin protein can expand, causing Huntington’s disease
Indels can cause frame shift, if indels are not multiples of three
Gene inversion
Whole genes are copied to offspring in reverse direction
Translocation
Whole genes can be deleted from one genome and inserted into another

7. Amino Acids General structure of amino acids
an amino group
a carboxyl group
α-carbon bonded to a hydrogen and a side-chain group, R
R determines the identity of particular amino acid
R: large white and gray
C: black
Nitrogen: blue
Oxygen: red
Hydrogen: white

9. AA Groups Classification of R groups Polar/nonpolar Acidic/basic
Polar share electron bonds unequally
O-H bond is polar: O is more electro-negative and bonding electrons are closer to O
C-H is nonpolar
Acidic/basic
Element
Electro-
negativty
Oxygen
3.5
Nitrogen
3.0
Sulfur
2.6
Carbon
2.5
Phosphorus
2.2
Hydrogen
2.1

10. Group 1: Nonpolar (hydrophobic)
Sometimes, Gly (G) is included because C-H bond is nonpolar

11. Group 2: Polar Side chains are electronically neutral (uncharged)
Ser (S), Thr (T), Cys (C), Asn (N), Gln (Q), Try (Y)
Asn (N) and Gln (Q) are consider derivatives of group 3 Asp (D) and Glu (E)

12. Group 3: Acidic Side chains have carboxyl group Asp (D) and Glu (E)
Side chains are negatively charged

13. Group 4: Basic His (H), Lys (K), Arg (R)
Side chain is positively charged
His (H), Lys (K), Arg (R)

14. Physico-Chemical Properties
Vol.
Alanine
Ala A 67
Arginine
Arg R
148
Asparagine
Asn N 96
Aspartic
Asp D 91
Cysteine
Cys C 86
Glutamine
Gln Q
114
Glycine
Gly G 48
Histidine
His H
118
Isoleucine
Ile I
124
Leucine
Leu L
Lysine
Lys K
135
Methionine
Met M
Phenyl.
Phe F
Proline
Prot P 90
Serine
Ser S 73
Threonine
Thr T 93
Tryptophan
Trp W
163
Tyrosine Y
141
Valine
Val V
105
Mean
109
Physico-chemical properties of AA determine protein structures
bioinformatics can be used via a pattern recognition
Properties
(1) Size in volume
Volume occupied by side groups is important (also for molecular evolution), and difficult to substitute a large AA for a small one
Van der Waals radius (volume until atoms are pushed to repulsion) is used to measure the volume of the sphere (in Å3)
W has 3.4 times the volume of G

15. (5) pH of isoelectric point of AA (pI)
(2) Partial Vol.
Measure expanded volume in solution when dissolved
(3) Bulkiness
The ratio of side chain volume to its length
Measure of average cross-sectional area of the side chain
Relevant to protein folding
(4) Polarity index
Electrostatic force acting on its surrounding at a distance of 10 Å
(5) pH of isoelectric point of AA (pI)
Acidic Asp and Glu have pI in 2-3: negatively charged at neutral pH due to ionization of COOH group to COO- -- need to put them in an acid solution to shift equilibrium and balance this charge (side chain is charged +)
Basic (Arg, Lys and His) has pI >7 (charged -)
All others have uncharged side chains (pl. in 5-6)

16. (6) Hydrophobicity (7) Surface area (8) Fraction of area
When molecules are dissolved in water, hydrogen-bonded structure is disrupted
Polar AA residues can form hydrogen bonds with water –hydrophilic
Non-polar that cannot form the bonds – hydrophobic
Polar disrupts the structure less than non-polar
Polar is usually at the exterior of a structure, non-polar, interior
Hydrophobicity (hydropathy) scale: estimate of difference in free energy of AA when buried in hydrophobic environment of the interior of a protein in water solution (+ for hydrophobic – costs free energy to take residue out of protein and put it in water)
(7) Surface area
Surface area of AA exposed (accessible) to water in an unfolded peptide chain and become buried when the chain folds
Relevant to protein folding
(8) Fraction of area
Fraction of the accessible surface area that is buried in the interor in a set of known crystal structures
Hydrophobic residues have a larger fraction

17. Red: acidic Orange: basic Green: polar Yellow: non-polar Vol. BulkpI
Hydro
Surf2
Frac
Alanine
Ala A 67
11.5
0.0
6.0
1.8
113
0.74
Arginine
Arg R
148
14.3
52.0
10.8
-4.5
241
0.64
Asparagine
Asn N 96
12.3
3.4
5.4
-3.5
158
0.63
Aspartic
Asp D 91
11.7
49.7
2.8
151
0.62
Cysteine
Cys C 86
13.5
1.5
5.1
2.5
140
0.91
Glutamine
Gln Q
114
14.5
3.5
5.7
189
Glu. Acid
Glu E
109
13.6
49.9
3.2
183
Glycine
Gly G 48
-0.4 85
0.72
Histidine
His H
118
13.7
51.6
7.6
-3.2
194
0.78
Isoleucine
Ile I
124
21.4
0.1
4.5
182
0.88
Leucine
Leu L
3.8
180
0.85
Lysine
Lys K
135
49.5
9.7
-3.9
211
0.52
Methionine
Met M
16.3
1.4
1.9
204
Phenyl.
Phe F
0.4
5.5
2.9
218
Proline
Prot P 90
17.4
1.6
6.3
-1.6
143
Serine
Ser S 73
9.5
1.7
-0.8
122
0.66
Threonine
Thr T 93
15.8
-0.7
146
0.70
Tryptophan
Trp W
163
21.7
2.1
5.9
-0.9
259
Tyrosine Y
141
18.0
-1.3
229
0.76
Valine
Val V
105
21.6
4.2
160
0.86
Mean
15.4
-0.5
175
Red: acidic
Orange: basic
Green: polar
Yellow: non-polar

18. “Universal” Genetic Code

19. Genetic Code 2

20. Properties Purine (A,G) is heavier than Pyrimidine (C,T)
Transition within a type (Purines or Pyrimidines) is more likely than Translation between types
All AAs have more than one codon, except for Met and Trp
Codons for an AA are clustered
Two codons for an AA – same in the first 2 positions and differ only by transition at the 3rd position
Four codons – differ only in the 3rd position
Six codons – form one four-codon box and one two-codon box

21. Genetic Code X X X X
Degeneracy is controlled by GC content of codons
G-C binding is stronger
First two bases (doublets) are GC – form four codon boxes (red X)
Doublets are AU – split boxes (blue X)
Doublets are mixed X X X X
Purine
2nd base is pyrimidine – four codon boxes, split otherwise
Larger purine at the 2nd position reduces binding at the 3rd position
A doublet forms a four-codon box, its ‘conjugate’ forms a split box
Conjugate – opposite size and opposite number of hydrogen bonds; A-C and G-U are conjugates

22. Genetic Code Five most hydrophobic – Phe, Leu, Ile, Met, Val
U at the 2nd position
Three most similar – Leu, Ile, Val
Single-base mutation at 1st position
Six most hydrophilic – His,Gln,Asn,Lys,Asp, Glu
A at the 2nd position
(Tyr is hydrophobic and has A in 2nd position)

23. Evolution of Genetic Code
From what the current Genetic Code became stable ?
Robin Knight

24. AA Substitutions 1978 – Dayhoff, Schwartz, Orcutt
Which AA substitutions are observed to occur when two homologous protein sequences are aligned ?
From aligned sequences of 71 families of closely related proteins (sharing more than 85% of sequences), tabulated 1572 substitutions
AA substitutions are accepted by natural selection occurs when
A gene undergoes a DNA mutation to translate to a different AA and does not significantly alter the gene function
The entire species adopts the change as a predominant form of the protein
Frequencies can represent expected mutation over short evolutionary distances
Called PAM (Point Accepted Mutation)
PAM unit corresponds to one AA change per 100 residues (1% divergence)

25. Dayhoff counting
Most freq. subs.: Glu to Asp (both acidic)

26. Protein Substitution Rates
Example
Six letters: I, K, L, Q, T, V
Seven sequences
Form an evolutionary tree
A: T L K K V Q K T
B: T L K K V Q K T
C: T L K K I Q K Q
D: I I T K L Q K Q
E: T I T K L Q K Q
F: T L T K I Q K Q
G: T L T Q I Q K Q

27. Protein Substitution Rates
Determine AAij
Count AA j being substituted by i
I K L Q T V I K L Q T V

28. Sub. Frequency to Score Matrix
AA mutation prob.
Mij : Prob. of original AA j mutating to AA i in one PAM distance
PAM distance: unit of evolutionary divergence in which 1% of AA's have changed between two protein sequences
Mij =  Aij /Ni (Ni count of amino acid i) -- normalized by the prob. of AA i occurring
Pij(t) : Prob. that a site has AA i at time t when it had j at time 0
Pij(dt) = Mij

29. Mutation Prob. Matrix Each entry is scaled by 105
Two most freq. substitutions are highlighted

30. Sub. Frequency to Score Matrix
2. Mutation Prob. Matrix to Log-Odds Scoring
qij : Prob. of aligning j to i
pi: prob. of observing AA i by chance
Odds – related to probability
prob = 0: odds = 0
prob = 0.5: odds = 1
prob =0.75: odds = 3 (75:25)
odds= prob/(1-prob) and prob = odds/(1+odds)
Log-odd: sij = 10*log(qij/ pi)
e.g., sED = 10*log( /0.062)
Can add log-odd scores

31. PAM matrix assumptions 1992 – Jones, Taylor, Thornton (JTT)
Most important assumption – each AA replacement is independent of previous mutations at the same position
Matrix can be extrapolated into predicted substitution fequencies at longer evolutionary distances
PAM1 multiplied by itself 100 times can represent what one would expect if there were 100 AA changes per 100 residues – PAM100
All sites are equally mutable independent of neighboring residues
No consideration of conserved blocks or motifs
Forces responsible for sequence evolution over shorter time span are identical to those for longer time spans
1992 – Jones, Taylor, Thornton (JTT)
59,190 substitutions in all sequences in Swiss-Prot

32. PAM1 matrix Mutation Prob. Matrix has Pij(t)
PAM1 matrix for related proteins with 1% mutation
= 99% identical between two sequences
For distantly related proteins, other PAM matrices are used by successively multiplying PAM1
PAM
% identity

33. PAM 250 matrix

34. Pairwise Alignment vs. PAM Distance
Two sequences 100 AAs
After 80 PAM distances (80 mutations), 50 AAs are different
After 250 hits, 20 AAs remain the same

35. PAM matrices Closely related: Human vs. Chimpanzee (100% AA identical)
Distantly related: HBA vs. HBB (43% AA identical)

36. BLOSUM S.Henikoff and J.G. Henikoff
Devised to perform best in identifying distant relationships
Based on BLOCKS database of aligned protein sequences
BLOcks Substitution Matrix
(# of observed pairs of AA at any position)/(# of pairs expected from the overall AA frequencies) is computed from regions of closely related proteins alignable without gaps
To avoid overweighting closely related sequences, groups of proteins with sequence identities higher than a threshold are replaced by either a single representative or a weighted average

37. BLOSUM 62 BLOSUM Threshold set at 62
Protein sequences sharing less than 62% identity
Default BLAST

38. BLOSUM62 Most popular diagonal Off-diagonal Score for exact match
W-W: score 11: because alignment of W between two sequences is rare
Off-diagonal
W (tryptophan) – Y (tyrosine): score 2
Positive score – occur more often than by chance, but replacement is not as good as if W is preserved (2 < 11) or if Y is preserved (2 < 7)
W – V (Valine): score -3

40. PAM vs. BLOSUM