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Lab Meeting June 7, 2001

Lab Meeting June 7, 2001. John Wrobel. HIV-1 Reverse Transcriptase. p66. heterodimer. p51. HIV-1 RT with DNA template. p66. p51. 66 kd subunit. thumb. fingers. RNaseH. connection. palm. Catalytic Site (aspartic acid triad). D110 D185 D186. p66 with DNA template.

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Lab Meeting June 7, 2001

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  1. Lab Meeting June 7, 2001 John Wrobel

  2. HIV-1 Reverse Transcriptase p66 heterodimer p51

  3. HIV-1 RT with DNA template p66 p51

  4. 66 kd subunit thumb fingers RNaseH connection palm

  5. Catalytic Site (aspartic acid triad) D110 D185 D186

  6. p66 with DNA template

  7. HIV-1 Reverse Transcriptase dNTP binding site nnRTi binding site RNaseH active site Image from Arnold Lab

  8. Fingers & Palm subdomainsin MMLV RT Active site Fingers Palm

  9. HIV-1 RT subunits(primary sequence) 1 85 323 438 120 151 243 560 fingers palm fingers palm thumb connection RNaseH p66 fingers palm fingers palm thumb connection p51

  10. HIV-1 RT expression vector

  11. Region of Mutagenesis in Fingers & Palm of HIV-1 RT Mutagenic cassette = P95 to E203

  12. Collection of HIV-1 RT single missense mutations 95 100 105 110 115 120 125 130 135 140 145 150 | | | | | | | | | | | | wild-type P H P A G L K K K K S V T V L D V G D A Y F S V P L D E D F R K Y T A F T I P S I N N E T P G I R Y Q Y N V L P sequence Conservative A A P A I R R A I P M N A E G F A I V E D E K R F P G Y P L S G V D D P T M K F E F D L V Substitutions T A N A A S S V T A I I S S A A Non- E N L V E F Q E N T L Q R G P V E F H V S L L D L F A G Y L W L D D L L I K H Y E H K G R R V N I N L D T A P L Conservative L L E L Q I M T V E G Y G V C T H W G A V N V E C V T L R K I A I L E F S C N T R Substitutions A Q A Q G R Y V F R A Y V I H V A E R R N I H K R Q D C F Q W Q C N T V S Q F C V V N T N Q L N L L F Q Q N 155 160 165 170 175 180 185 190 195 200 | | | | | | | | | | wild-type Q G W K G S P A I F Q S S M T K I L E P F R K Q N P D I V I Y Q Y M D D L Y V G S D L E I G Q H R T K I E sequence Conservative A F A S T M Y E I S R L V D A Y K R E A E V M F E F I E E V F I A A E I D L E R A V D Substitutions T S V V M S L L L N N M L P N V S M L V V T P Non- P R G I E L R L R C P R C K I T S F V H C S T K K L H T D F H P N K V A F C A R F Y F K T V R Y T I N K V Conservative H V C E R K L L C I T T S L I T Q P T R V R F T S R C G H W G E Y V G K W L N I K I R G Substitutions R N L F A I N R F F Q R L G N R S A F S H D Y G S G V R R H P S R Q A Q N H N N N Q I L N K H Y N A Q F S N

  13. Analysis of 366 HIV-1 RT mutants (part 1)

  14. Analysis of 366 HIV-1 RT mutants (part 2)

  15. Problems with contact definition • How to define a contact unambiguously? • How many contacts does a residue make? • Analysis of 3D structures requires 3D definition of contacts • Delaunay tessellation provides a unique way to define FOUR nearest neighbors in 3D as vertices of tetrahedra

  16. Voronoi Tessellation Voronoi tessellationpartions space into convex polytopes calledVoronoi polyhedra Proteins Voronoi polyhedronis the region of space around an atom, such that each point of this region is closer to the atom than to any other atom

  17. Delaunay Simplex A group of 4 atoms whose Voronoi polyhedra meet at a common vertex forms aDelaunay simplex Delaunay tessellationof a protein structure generates an aggregate of space-filling non-overlapping irregular tetrahedra

  18. Delaunay Simplices 2D Delaunay Simplices = triangles 3D Delaunay Simplices = tetrahedra

  19. Voronoi/Delaunay Tessellation in 2D Delaunay simplex isdefined by points, whose Voronoi polyhedra havecommon vertex Delaunay simplex is always a triangle in a 2D space and a tetrahedron in a 3D space Voronoi Tessellation Delaunay Tessellation

  20. Defining nearest neighbors Each amino acid residue represented by a single point (aC) Vertices of each simplex objectively define 4 nearest aC atoms & therefore 4 nearest neighbor residues

  21. Differences Voronoi polyhedramay differ topologically (they may have different number of faces & edges) Delaunay simplicesare always topologically equivalent (tetrahedra in 3D space)

  22. 5 Classes of Delaunay Simplices {4} = all residues of simplex are consecutive in protein seq {3,1} = 3 residues consecutive, 4th is distant in seq {2,2} = 2 pairs of consecutive residues are distant in seq {2,1,1} = 2 residues consecutive & 2 other residues are distant from the first 2 & from each other {1,1,1,1} = all 4 residues are distant from each other

  23. Classification of Delaunay simplices bysequential proximity

  24. Database • Dataset of unique protein structures identified (Pro Sci 3, 522) • This dataset contains 322 protein chains (66,852 amino acids) • with high crystallographic resolution that do not have apparent • structural similarity and carry low sequence identity (25%) • Tessellation of the dataset generates 387,880 simplices

  25. Statistical analysis of Delaunay Simplex Composition • Quadruplet Composition Types • 204 = 160,000 • Geometrical Description of Tetrahedra: sequence order independence of composition • Theoretical Number of Quadruplets is reduced to 8855

  26. Compositional Propensities of Delaunay Simplices  observed freq  expected freq Ratio: 1  non-specific to folding > 1 some forces that bring them together, some specificity

  27. Equation q = log-likelihood factor ijkl = amino acid residues qijkl = q for a given quadruplet (likelihood of finding 4 particular residues in a simplex) fijkl = observed freq of occurrence of a given quadruplet

  28. Log-likelihood of amino acid quadruplets with different compositions 1 CCCC 3.081003 2 CCCY 2.13004 3 CCHH 1.960814 4 CCCG 1.782267 5 CCCH 1.742759 6 CCCW 1.724275 7 CCCS 1.724275 8 CCCQ 1.657329 Log-likelihood ratio 8343 CDDL -0.90166 8344 IRRV -0.90217 8345 AEYY -0.90535 8346 KKRV -0.95081 8347 CKRS -0.96133 8348 CEKP -0.98433 8349 HKKS -0.98472 8350 CGLR -1.14737

  29. Plot reveals highly non-random distribution For some quadruplets observed frequencies are orders of magnitude higher (or lower) than expected from random model

  30. PROCAM Protein Core Alignment Map View with Netscape

  31. My Project Goal: Combine protein chemistry & protein evolution Evolution of retroviral RTs Are hydrophobic cores found in same place? (Procam) Keeping track of aa residues among retroviral RTs conversion file (Microsoft Access)

  32. Retroviral Tree HSRV (spumavirus) MMLV (mammalian C-type) BLV (HTLV/BLV) RSV (avian C-type) MMTV (B-type) MPMV (D-type) HIV-1 (lentivirus)

  33. Region of Eickbush alignment fingers palm fingers palm thumb connection RNaseH HIV E44 L234 MMLV L82 L273

  34. Eickbush region in HIV-1 RT

  35. Eickbush region in HIV-1 RT

  36. Eickbush region in MMLV RT

  37. HIV-1 conserved tetrahedra

  38. HIV-1 conserved tetrahedra (> 0.9)

  39. HIV-1 conserved tetrahedra (> 0.9)

  40. HIV-1 conserved tetrahedra (0.6 to 0.9)

  41. HIV-1 conserved tetrahedra (0.3 to 0.6)

  42. HIV-1 conserved tetrahedra (0 to 0.3)

  43. HIV-1 conserved tetrahedra (negative) Kinimage

  44. MMLV conserved tetrahedra

  45. MMLV conserved tetrahedra (> 0.9)

  46. MMLV conserved tetrahedra (> 0.9)

  47. MMLV conserved tetrahedra (0.6 to 0.9)

  48. MMLV conserved tetrahedra (0.3 to 0.6)

  49. MMLV conserved tetrahedra (0 to 0.3)

  50. MMLV conserved tetrahedra (negative) Kinimage

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