title: Solid-state NMR assessment of enzyme active center structure under nonaqueous conditions.
abstract: By using solid-state NMR spectroscopy, the integrity of the active center of alpha-chymotrypsin was investigated under a variety of nonaqueous conditions. Specifically, 13C cross-polarization/magic angle spinning NMR was used to analyze the ability of alpha-chymotrypsin to stabilize a transition state intermediate analog after freezing, drying, and addition of organic solvents (both anhydrous and hydrated) to the resultant powder. Lyophilization disrupted 42 +/- 5% of the active centers; it was determined that this occurred during drying, as opposed to freezing. Seven anhydrous solvents caused 0-50% additional disruption, which occurred immediately on addition of the solvent to the enzyme powder. The extent of structural integrity loss correlated with the solvent hydrophobicity, indicating that further dehydration, i.e. stripping of water retained by the enzyme during lyophilization, was the cause. Enzyme samples prepared with lyoprotecting additives, sucrose and ammonium sulfate, exhibited varying degrees of stabilization against the drying step of lyophilization. Moreover, when hydrophilic anhydrous solvents, which had the highest propensity to strip bound water, were added to the resultant enzyme powders, no additional damage occurred.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
authors.forename: P A
authors.initials: PA
authors.lastname: Burke
authors.forename: R G
authors.initials: RG
authors.lastname: Griffin
authors.forename: A M
authors.initials: AM
authors.lastname: Klibanov
mesh_terms: D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012997:Solvents; D013395:Sucrose / Q000737:chemistry; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000737:chemistry
chemical_list: D012997:Solvents; D014108:Tosylphenylalanyl Chloromethyl Ketone; D013395:Sucrose; D002918:Chymotrypsin; D000645:Ammonium Sulfate