Craescu C T, Mispelter J, Blouquit Y
INSERM U.91, CNRS UA607, Hôpital Henri Mondor, Créteil, France.
Biochemistry. 1990 Apr 24;29(16):3953-8. doi: 10.1021/bi00468a023.
Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.
血红蛋白圣芒德（β N102Y）是一种低亲和力突变体，其取代位点位于对四级结构敏感的α1β2界面。在成人血红蛋白中，β链上的Asn102有助于配体结合（R）态的稳定性，与α链上的Asp94形成氢键。通过一维和二维核磁共振（NMR）光谱研究了单羧化血红蛋白圣芒德中酪氨酸取代天冬酰胺后四级和三级结构的扰动。对1H2O中配体结合和未结合样品的一维NMR光谱分析表明，血红蛋白圣芒德的R态和T态四级结构均与血红蛋白A中的相应结构不同。在酸性pH条件下，突变血红蛋白的单羧化形式中，我们观察到一种R型氢键消失，出现了一种新的氢键，其质子共振类似于脱氧T标记物。使用二维NMR方法，并基于先前对单羧化血红蛋白A的研究结果，我们在有或没有强变构效应剂肌醇六磷酸存在的情况下，获得了pH 5.6时单羧化血红蛋白圣芒德光谱中的大量共振归属。这使我们能够表征由四级结构修饰引发的三级构象变化（相对于配体结合的正常血红蛋白）。观察到的结构变化局限于血红素口袋区域，但涉及α和β两个亚基。其中大多数位于C、F、G和FG片段，可能直接由侧链取代引起，而其他一些，如β链上的Leu141，只能通过远程相互作用来解释。
