Twenty-five years of Research on Cytochromes P450 2B: To ...

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Conformational Plasticity <strong>of</strong> P4502B Enzymes• X-ray crystallography <strong>of</strong> rabbit P450 2B4• Hydrogen-deuterium mass exchangestudies <strong>of</strong> 2B4• X-ray crystallography <strong>of</strong> human P450 2B6
• protein engineering:highly concentrated proteinsolution(+ salt, detergent, DTT,EDTA, buffer . . .)+Control <strong>of</strong> Stability/Solubility1 10 20 30 4902B4 MALLLAVLLA FLAGLLLLLF RGHPKAHGRL PPGP . . . AR2B4dH MA KKTSSKGKL PPGP . . . ARHHHHUnderline indicates alteration from the native sequence.• salt• detergentprecipitant(EtOH, Na citrate pH 5.5)Crystallizationprotein + precipitantprecipitant sitting dropvapor diffusion• The engineering and purification approaches elaborated for P4502C5 enable 2B4 crystallization.
Page 3 and 4: Goals of Predictiv
Page 5 and 6: Species, Strain, and IndividualDiff
Page 7 and 8: 14 C (cpm)Halpert, J. (1981). Coval
Page 9 and 10: Halpert, J., Jaw, J.-Y., Balfour, C
Page 11: 245-HCB Metabolism(% of</st
Page 15: % P450 ExtractedScott, E.E., Spatze
Page 19 and 20: Location of Active
Page 21 and 22: Comparison of 2B4
Page 23 and 24: P450 PlasticityDefinition• Involv
Page 25 and 26: Comparison of Five
Page 27 and 28: DX-MS Setup (Exchange)• Remove al
Page 29 and 30: B’-C RegionF-G Cassette2B4dH Exch
Page 31 and 32: CYP2B4:Ticlopidine Complex Ribbon D
Page 33 and 34: Structure of Ticlo
Page 36 and 37: Molecular ReplacementE301T302• Ma
Page 38 and 39: Properties of Huma
Page 40 and 41: Approaches to EnableCrystallization
Page 42 and 43: Structural Alignment of</st
Page 44 and 45: Summary1. P450 2B4 exhibits remarka
Page 46 and 47: Multiplicity of P4
Page 48 and 49: Halpert, J.R. (1988). Multiplicity
Page 50 and 51: Complications Presented by CYP3A4
Page 52 and 53: Multiple Conformers as an Alternati
Page 54 and 55: • Lack of Substr
Page 56 and 57: Lessons Learned from Colleagues•

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