Assessing Native and Non-native Conformational States of a Protein by Methylene Carbene Labeling: The Case of Bacillus licheniformis -Lactamase

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• 作者：Daniela B. Ureta ; Patricio O. Craig ; Gabriela E. Gó ; mez ; Jos&eacute ; M. Delfino
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：December 18, 2007
• 年：2007
• 卷：46
• 期：50
• 页码：14567 - 14577
• 全文大小：241K
• 年卷期：v.46,no.50(December 18, 2007)
• ISSN：1520-4995

文摘

Much knowledge of protein folding can be derived from the examination of the nature andsize of solvent-exposed surfaces along conformational transitions. We exploit here a general photochemicalmodification with methylene carbene of the accessible surface area (ASA) of the polypeptide chain. Labelingof Bacillus licheniformis -lactamase (BL-L) with 1 mM ³H-diazirine yielded 8.3 × 10^-3 mol CH₂/molprotein, in agreement with the prediction for an unspecific surface labeling phenomenon. The unfoldedstate U in 7 M urea was labeled 60% more than the native state N. This result lies well below the incrementof ASA expected from theoretical estimates and points to the presence of residual organization in state Uand/or of cavities or crevices favoring the partition of the reagent in state N. A partially folded state I wasdemonstrated from two sequential transitions occurring at 1.5-3.0 M and 3.5-6.5 M urea. This techniqueshows a close correlation with optical probes most sensitive to changes in tertiary structure, a statementsupported by the fact that the largest change occurs along the N-I portion of the N-I-U transition andalong the acid pH-induced N-A transition. In the latter case, state A is labeled 70% more than state N,an increment consistent with the loosening of tight interactions in the core of the protein. Fragmentationof labeled BL-L into peptides provides a sequential map of solvent accessibility. Thus, amino acid residuespertaining to the -loop and to helices 5 and 6 line the major cavity of the protein, that is big enoughto lodge the diazirine reagent. Methylene labeling, by introducing an original (and perhaps unique)experimental measurement of ASA, enlightens subtle aspects of complex transitions and makes possiblea comparative structural characterization of the native as well as non-native states.