Use of organic cosmotropic solutes to crystallize flexible proteins: application to T7 RNA polymerase and its complex with the inhibitor T7 lysozyme.
We have crystallized, using several approaches that may be of general interest, T7 RNA polymerase (T7RP) and the T7 RNA polymerase-T7 lysozyme complex (T7RPL) in forms suitable for structure determination by X-ray crystallography. A series of polyhydric alcohols, sugars, amino and methylamino acids, compounds known to stabilize protein structure, were found to be critical for both crystallization and subsequent improvement of the crystal's diffraction resolution. Moreover, optimal crystallogenesis was achieved through an unconventional "reverse" vapor diffusion sitting drop method that is suitable for proteins that are insoluble at low ionic strength.T7RP has been crystallized in an orthorhombic form (I), space group P222, with cell parameters a=220 A, b=205 A, c=67 A and a monoclinic form (II), space group P21, with cell parameters a=229 A, b=205 A, c=70 A, beta=106 degrees. Crystal form I diffracts X-rays to 3.5 A and form II to 6.0 A. Three and six copies of the polymerase are predicted to be in the asymmetric unit forms I and II, respectively. Three monoclinic crystal forms of the T7RPL complex have been obtained in space group C2. Form I has cell parameters a=320 A, b=93 A, c=229 A, beta=129 degrees, form II has parameters a=293 A, b=93 A, c=68 A, beta=93 degrees, and form III has parameters a=270 A, b=93 A, c=63 A, beta=103 degrees. Crystal form I diffracts synchrotron wiggler radiation to 3.2 A and form III to 2.8 A. Calculations of crystal density imply three or four copies of the complex in form I and one copy in the asymmetric unit of forms II and III.
Jeruzalmi
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Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA.
Steitz
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D000596:Amino Acids; D017123:Bacteriophage T7 / Q000201:enzymology*; D001622:Betaine; D002241:Carbohydrates; D002331:Carnitine; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004791:Enzyme Inhibitors; D008744:Methylamines; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry* / Q000494:pharmacology; D013402:Sugar Alcohols; D014764:Viral Proteins
D000596:Amino Acids; D002241:Carbohydrates; D004791:Enzyme Inhibitors; D008744:Methylamines; D013402:Sugar Alcohols; D014764:Viral Proteins; D001622:Betaine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009238:N-Acetylmuramoyl-L-alanine Amidase; D002331:Carnitine