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Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

¹ Department of Chemistry, Loyola University, New Orleans, Louisiana 70118, USA
² Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA
³ Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA
⁴ Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059, USA

Reprint requests to: W.F. Walkenhorst, Chemistry Department, Loyola University New Orleans, Box 5, 6363 St. Charles Avenue, New Orleans, LA 70118, USA; e-mail: walken{at}loyno.edu; fax: (504) 865-3270.

Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid-denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly ¹⁵N-labeled H124L SNase was monitored as a function of refolding time by heteronuclear two-dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen-bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the ß-barrel and to a lesser degree in the -helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a ß-hairpin formed by strands 2 and 3 of the ß-barrel domain and a single site near the C-terminus. The results indicate that formation of stable hydrogen-bonded structure in a core region of the ß-sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.

Keywords: Staphylococcal nuclease; pulse labeling; hydrogen exchange; NMR; protein folding

Abbreviations: C_m, midpoint of a GuHCl denaturation transition • DCl, ²HCl; D₂O, ²H₂O • GuHCl, guanidine hydrochloride • H124L SNase, staphylococcal nuclease with a histidine-to-leucine substitution at position 124 (sequence equivalent to the staphylococcal nuclease isolated from the V8 strain of Streptococcus aureus) • m, slope of the GuHCl denaturation transition in units of kcal mol^-1 M^-1 • HSMQC, heteronuclear single-quantum, multiple-quantum correlation • pdTp, deoxythymidine-3`,5`-bisphosphate • pH*, pH of a sample dissolved in D²O as determined by an uncorrected glass electrode measurement • Pro^-, mutant of SNase with P11A, P31A, P42A, P47G, P56A, and P117G substitutions • SNase, wild-type staphylococcal nuclease with a sequence equivalent to that isolated from the Foggi strain of S. aureus).

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