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 beta strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI).
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 beta strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI).
复制标题
用于检测相邻主链肽酰胺相关氢交换的碳 13 NMR 方法及其在链霉菌枯草杆菌蛋白酶抑制剂 (SSI) 疏水核心内五个反向平行 β 链氢交换中的应用。
DOI：
10.1021/bi050467s
复制
发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
Kainosho,Masatsune
中科院分区：
文献类型：
--
作者：
Uchida,Kenichi;Markley,JohnL;Kainosho,Masatsune
一种新的方法来监测质子-氘（H/D）交换的骨干酰胺是基于观察的H/D同位素效应的13 C NMR信号从肽羰基。羰基13 C信号的线形受两个相邻酰胺：HNi+1（β位）和HNi（γ位）处的不同H/D占据的影响。在75.4 MHz的碳频率下，13 C信号上的H → D同位素位移对于β位点的交换约为5−7 Hz，对于γ位点的交换约为2 Hz或更少。由于在两个网站的效果是加性的，一个特定的羰基共振的线形的时间依赖性可以报告不仅在个别网站的交换率，但也双交换的水平。因此，可以分析数据以确定两个位点的相关交换率（kc）和程度（Xβγ）。我们应用这种方法研究了Streptomycessubtilisinhibitor（SSI）中几个相邻残基的氢交换对pH的依赖性。产生了两种选择性标记的SSI蛋白：一种在所有缬氨酰残基处具有选择性13 C '标记，另一种在所有亮氨酰残基处具有选择性13 C'标记。这使得直接观察一维13 C NMR的选择羰基信号与缓慢交换酰胺在theiandi+ 1位置的残基。研究的残基位于α螺旋和五链反平行β折叠中。在不同pH* 值下制备两种标记蛋白质的样品，并在将样品从H2O转移到2 H2O之前和之后的不同时间在50 °C下收集13 C NMR光谱。研究的大多数慢交换酰胺是分子内氢键供体。与先前的研究一致，结果表明，蛋白质中酰胺氢的交换速率不仅受氢键作用的影响，还受其他因素的影响。例如，Thr 34的酰胺氢交换迅速，即使它是分子内氢键供体。在几乎整个研究的pH范围内，表观非相关交换速率（kβ和k γ）与[OH-]成正比，两个相邻位点的表观相关交换速率（kc）与[OH-]2大致成正比。这使得我们能够提取与pH无关的交换速率（kβ°、kγ°和kc °）。在所有可以测量相关交换的情况下，所观察到的X βγ的S形pH依赖性可以从导出的pH无关速率大致复制。
A novel method for monitoring proton−deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the13C NMR signals from peptide carbonyls. The line shape of the carbonyl13Cisignal is influenced by differential H/D occupancy at the two adjacent amides: the HNi+1(β site) and the HNi(γ site). At a carbon frequency of 75.4 MHz, the H → D isotope shifts on the13C signal are about 5−7 Hz for exchange at the β site and 2 Hz or less for exchange at the γ site. Because 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 (kc) and degree of correlated exchange (Xβγ) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues inStreptomycessubtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective13C‘ labeling at all valyl residues and one with selective13C‘ labeling at all leucyl residues. This permitted the direct observation by one-dimensional13C NMR of selected carbonyl signals from residues with slowly exchanging amides at theiandi+ 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, and13C NMR spectra were collected at 50 °C prior to and at various times after transferring the sample from H2O to2H2O. 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 (kβandkγ) were proportional to [OH-] and the apparent rates of correlated exchange at two adjacent sites (kc) were roughly proportional to [OH-]2. This enabled us to extract the pH-independent exchange rates (kβ°,kγ°, andkc°). In all cases in which correlated exchange could be measured, the observed sigmoidal pH dependence ofXβγcould be replicated roughly from the derived pH-independent rates.