Side-chain mobility of the beta-lactamase A state probed by electron spin resonance spectroscopy.
beta-Lactamase from Bacillus licheniformis forms a stable compact intermediate state at low pH and moderate salt concentration (the A state), with properties consistent with a molten globule. A single cysteine residue was introduced into this class A beta-lactamase by site-directed mutagenesis at position 166. A spin label was attached to the thiol of this cysteine residue via a disulfide bond as a probe of the side-chain mobility. The mutant protein and the spin-labeled derivative exhibited similar conformational properties to the wild-type enzyme at acidic pH. The A state induced by chloride or trichloroacetate (TCA) anions was characterized by circular dichroism and esr. The A state at pH 0.5 (0.32 M HCl), or at pH 2 in the presence of 8 mM TCA or 0.4 M Cl-, had comparable amounts of secondary structure to the native state but lacked significant tertiary structure, as judged by the lack of near-UV circular dichroism. Analysis of the esr spectral line widths showed that the mobility of the spin label in the A state was similar to that in the native state and much less mobile than in the unfolded state, indicating significant constraints on the side-chain mobility in this region of the molecule in the A state. The implications of this finding to the structure of the A state are discussed.
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
L J
LJ
Calciano
W A
WA
Escobar
G L
GL
Millhauser
S M
SM
Miick
J
J
Rubaloff
A P
AP
Todd
A L
AL
Fink
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D002942:Circular Dichroism; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels; D013816:Thermodynamics; D014508:Urea; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D013113:Spin Labels; D014508:Urea; D001618:beta-Lactamases; D003545:Cysteine