Carbon-13 NMR Method for the Detection of Correlated Hydrogen Exchange at Adjacent Backbone Peptide Amides and Its Application to Hydrogen Exchange in Five Antiparallel β Strands within the Hydrophobic Core of Streptomyces Subtilisin Inhibitor (SSI)

Abstract

A novel method for monitoring proton-deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the 13 C NMR signals from peptide carbonyls. The line shape of the carbonyl 13 C i signal is influenced by differential H/D occupancy at the two adjacent amides: the H N i+1 (β-site) and the H N i (γ-site). At a carbon frequency of 75.4 MHz, the H→D isotope shifts on the 13 C signal are about 5-7 Hz for exchange at the β-site and 2 Hz or less for exchange at the γ-site. Since the effects at the two sites are additive, the time-dependence of the line shape of a particular carbonyl resonance can report not only the exchange rates at the individual sites, but also the level of dual exchange. Therefore, the data can be analyzed to determine the rate (k c ) and degree of correlated exchange (X βγ ) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues in Streptomyces subtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective 13 C′ labeling at all valyl residues and one with selective 13 C′ labeling at all leucyl residues. This permitted the direct observation by one-dimensional 13 C NMR of selected carbonyl signals from residues with slowly exchanging amides at the i and i+1 positions. The residues investigated were located in an α-helix and in a five-stranded antiparallel β-sheet. Samples of the two labeled proteins were prepared at various pH*values, and 13 C NMR spectra were collected at 50 °C prior to and at various times subsequent to transferring the sample from H 2 O to 2 H 2 O. Most of the slowly exchanging amides studied were intramolecular hydrogen bond donors. In agreement with prior studies, the results indicated that the exchange rates of the amide hydrogens in proteins are governed not only by hydrogen bonding, but also by other factors. For example, the amide hydrogen of Thr34 exchanges rapidly even though it is an intramolecular hydrogen bond donor. Over nearly the whole pH range studied, the apparent rates of uncorrelated exchange (k β and k γ ) were proportional to [OH − ], and the * To whom correspondence should be addressed. Telephone: +81-426-77-2544+81-426-77- . Fax: +81-426-77-2544 NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 27. Published in final edited form as:Biochemistry. . This enabled us to extract the pH-independent exchange rates (k β°, k γ°, and k c°) . In all cases in which correlated exchange could be measured, the observed sigmoidal pH-dependence of X βγ could be replicated roughly from the derived pH-independent rates. KeywordsStreptomyces subtilisin inhibitor; SSI; carbonyl 13 C NMR; stable isotope assisted NMR; H/D exchange; amide hydrogen; isotope shift; β-shift; γ-shift; correlated hydrogen exchangeThe internal fluctuations of protein molecules are deeply related to their structure and stability and contribute to their functions. Protein fluctuations have been ...

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“…The carbonyl chemical shift is affected by deuterium substitution of its neighboring amide positions [86,87]. This deuterium-induced isotope shift on 13 C signals ($0.1 ppm) was observed even at 52°C for the H(C)CO peaks of the Fc dissolved in a buffer (pH 6.0) containing a 1:1 mixture of H 2 O and D 2 O, indicating a slow hydrogen-deuterium exchange of the acetamide groups (Fig.…”

Cooperative α‐helix unfolding in a protein–DNA complex from hydrogen–deuterium exchange

“…For instance, 1D difference NOE NMR on a partially exchanged sample (Wagner 1980; Roder et al 1985) indicated correlated exchange in the central part of an antiparallel β‐sheet in the bovine pancreatic trypsin inhibitor (BPTI), and H–D exchange monitored by mass spectroscopy revealed correlated exchange in lysozyme (Miranker et al 1993), in the native turkey ovomucoid third domain (Arrington et al 1999; Arrington and Robertson 2000) and in cytochrome c associated with negatively charged lipid membranes (Pinheiro et al 2000). Recently, Uchida et al (2005) reported an elegant method based on the observation of H/D isotope effects from neighboring amide hydrogens on the carbonyl 13 C chemical shift to detect correlated exchange, and applied it to the Streptomyces subtilisin inhibitor (SSI). In this method, the correlated exchange effects can be directly observed in 1D NMR spectra.…”

“…Approaching the EX1 limit to maximize the sensitivity of the observable k obs on these opening motions (see k op ), therefore, is a prerequisite for studies on the degree of correlation between the H–D exchange of neighboring amide protons. While local or global unfolding processes were shown to be involved in the mechanism of H–D exchange in various systems (Kim and Woodward 1993; Mayo and Baldwin 1993; Bai et al 1995; Loh et al 1996; Sivaraman et al 2001; Qu and Bolen 2003), correlated exchange of neighboring H N has rarely been demonstrated experimentally (Wagner 1980; Roder et al 1985; Pinheiro et al 2000; Uchida et al 2005).…”

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“…1,[19][20][21] In contrast to the 1 H detection experiments, the signal dispersion in the 13 C chemical shifts and the narrower linewidths are advantages of the 13 C direct observation approach for relatively large proteins. An NMR experiment using direct 13 C observation may also yield useful structural information, which cannot easily be obtained by the other methods.…”