“The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and
-
Upload
vivien-cooper -
Category
Documents
-
view
14 -
download
1
description
Transcript of “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and
![Page 1: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/1.jpg)
“The instructions for assembling every organismon the planet--slugs and sequoias, peacocks andparasites, whales and wasps--are all specified in DNAsequences that can be translated into digital information and stored in a computer for analysis. As a consequence of this revolution, biology in the 21st century is rapidly becoming an information science...
...hypotheses will arise as often in silico as in vitro.”
Eric Lander, Science 287 (5459), 1777-1782
![Page 2: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/2.jpg)
![Page 3: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/3.jpg)
The Problem …analysis of native state assembly of proteins.
• Protein function and folding are highly cooperative processes,
• Amino acids that interact in these processes can be close, or relatively distant in the 1o structure,
– identifying interacting residues in active sites, or identifying interacting residues that yield discrete 3o structure is difficult,
– these interactions are not obvious by scanning primary sequence.
![Page 4: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/4.jpg)
A Partial Solution
• Mutational analysis,
– clone the gene,
– alter the DNA sequence that codes for specific residues,
– express the gene,
– check for function or conformational fidelity.
Labor intensive. Doesn’t indicate residue interactions.
![Page 5: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/5.jpg)
A Better SolutionThermodynamic Mutant Cycling Analysis
More later…but briefly…
Double mutation analysis,
– used to determine if two different residues (or peptide fragments) interact.
Labor intensive.
Presently impossible to accomplish on a large scale.
![Page 6: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/6.jpg)
A Bioinformatic Alternative…...let Evolution do the dirty work.
Multiple Sequence Alignment (globins)
![Page 7: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/7.jpg)
“Entropy” in a MSA…the key to this paper.
• Think of amino acids as parts of a system that follows the rules of thermodynamics,
– if there were no constraints, amino acid frequency and distribution would tend to randomness,
– however, natural selection constrains primary sequence in living systems.
![Page 8: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/8.jpg)
MSA and Entropy
![Page 9: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/9.jpg)
Genomic SequencesDNA Sequence:Reagent for the 21st Century
![Page 10: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/10.jpg)
PDZ Domains (n = 274)
“Model” Protein Domain Family
• Evolutionarily conserved, especially in tertiary structure,
– C atoms: root mean square deviation = 1.4 angstroms*,
• More diverged in sequence homology,
– averaging 24% AA sequence similarity.
• *Four high resolution crystal structures of distantly related family members.
Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and Zonula occludens-1 protein (zo-1)
![Page 11: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/11.jpg)
Structural Classification of Proteinsdomains
Google: SCOP
Pfam
PDZ domains are found in diverse signaling proteins in bacteria, yeasts, plants, insects and vertebrates. They bind either the carboxyl-terminal sequences of proteins or internal peptide sequences
PDZ domains consist of 80 to 90 amino acids comprising six beta-strands (betaA to betaF) and two alpha-helices, A and B, compactly arranged in a globular structure. Peptide binding of the ligand takes place in an elongated surface groove as an antiparallel beta-strand interacts with the betaB strand and the B helix. The structure of PDZ domains allows binding to a free carboxylate group at the end of a peptide through a carboxylate-binding loop between the betaA and betaB strands.
![Page 12: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/12.jpg)
Don’t Sweat the Formulas!
…English Translation: a measure of conservation can be made by comparing the frequency of amino acids in the column of a MSA, to a randomly filled column…
…expressed as a change in free energy.
![Page 13: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/13.jpg)
Figure 1A
Black: amino acid frequency in a database of 36,498 proteins.
Gray: amino acid frequency in a database of 274 PDZ domains.
![Page 14: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/14.jpg)
PDZ domain AA 76
• AA 76 is known to be important in determining ligand specificity,
- S/T - X- V/I - COO- - or - - F/Y - X- V/A - COO-
Antepenultimate AA in the ligand.
![Page 15: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/15.jpg)
Figure 1B,CPDZ MSA Gstat
Highly conserved.
Poorly conserved.
Gstat = 3.83 kT*
Gstat = 0.1 kT*
![Page 16: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/16.jpg)
Figure 1D
76
99
![Page 17: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/17.jpg)
Figure 1E, F
![Page 18: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/18.jpg)
Coupled Sites?
…English Translation: you change the MSA by removing a subset of peptides that have similar (or identical) amino acids in a specific column…
…if the amino acid in the original column interacts with another part of the peptide, you might expect to see a change in Gstat (Gstat ) in another column of the new MSA.
![Page 19: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/19.jpg)
Perturbing the MSA…extract subsets of low-entropy alignments.
Re-calculate Gstat in the new MSA, look
for columns that had a change in
Gstat.
![Page 20: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/20.jpg)
AA 76
– removed all of the peptides that had a histidine at AA 76 in the MSA,
• Calculated the change in Gstat (Gstat) at all positions.
![Page 21: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/21.jpg)
Figure 2 B AA 76
AA 34
AA 63
![Page 22: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/22.jpg)
Figure 2C-F
33, 34, 80, 84local
29, 26other side of ligand binding
66, 57, 51unexpected
![Page 23: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/23.jpg)
in Silica, So Far, So What?
Show me the money...
![Page 24: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/24.jpg)
Fig. 3
Statistical Gstat
Experimental Gstat
H76Y
![Page 25: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/25.jpg)
FRET Fluorescence Resonance Energy Transfer
![Page 26: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/26.jpg)
Mutant Cycling Analysis (General)…with FRET (Fluorescence Resonance Energy Transfer)
ratio m1
ratio m1:m2
If not equal, then sites are coupled.
Please Note: this was a general presentation, see slide 33 for the application used in this paper.
![Page 27: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/27.jpg)
Fig. 3
?
Fig. D: What is it, why is it included?
![Page 28: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/28.jpg)
Figure 4
Attempt to map connectivity through the peptide.
Also performed analysis on POZ domain.
![Page 29: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/29.jpg)
Conclusion
With growing sequence data from evolutionary distant genomes, the mapping
of energetic connectivity for many fold families should be a realistic goal.
![Page 30: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/30.jpg)
Figure 4
![Page 31: “The instructions for assembling every organism on the planet--slugs and sequoias, peacocks and](https://reader036.fdocuments.in/reader036/viewer/2022070400/56813590550346895d9cffd2/html5/thumbnails/31.jpg)
k (wt:wt) x k (mut:mut)
k (wt:mut) x k (mut:wt)
Coupling Coefficient(Mutant Cycling Analysis)
coupling coefficient =
...if there is no coupling, then the coupling coefficient would approach unity.