Dass Rupashree, Corlianò Enrico, Mulder Frans A A
Department of Chemistry, and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, 8000, Aarhus, Denmark.
Department of Chemistry, University of Florence, 50019, Sesto Fiorentino, (FI), Italy.
Chemphyschem. 2019 Jan 21;20(2):231-235. doi: 10.1002/cphc.201801044. Epub 2018 Dec 18.
NMR spectroscopy is a pivotal technique to measure hydrogen exchange rates in proteins. However, currently available NMR methods to measure backbone exchange are limited to rates of up to a few per second. To raise this limit, we have developed an approach that is capable of measuring proton exchange rates up to approximately 10 s . Our method relies on the detection of signal loss due to the decorrelation of antiphase operators 2N H by exchange events that occur during a series of pi pulses on the N channel. In practice, signal attenuation was monitored in a series of 2D H(CACO)N spectra, recorded with varying pi-pulse spacing, and the exchange rate was obtained by numerical fitting to the evolution of the density matrix. The method was applied to the small calcium-binding protein Calbindin D , where exchange rates up to 600 s were measured for amides, where no signal was detectable in N- H HSQC spectra. A temperature variation study allowed us to determine apparent activation energies in the range 47-69 kJ mol for these fast exchanging amide protons, consistent with hydroxide-catalyzed exchange.
核磁共振光谱法是测量蛋白质中氢交换速率的关键技术。然而，目前可用的测量主链交换的核磁共振方法仅限于每秒几的速率。为了提高这个限度，我们开发了一种能够测量高达约10 s的质子交换速率的方法。我们的方法依赖于检测由于在N通道上一系列π脉冲期间发生的交换事件导致的反相算符2NH去相关而引起的信号损失。在实践中，在一系列二维H(CACO)N光谱中监测信号衰减，这些光谱以不同的π脉冲间隔记录，并且通过对密度矩阵的演化进行数值拟合来获得交换速率。该方法应用于小钙结合蛋白钙结合蛋白D，其中酰胺的交换速率高达600 s，在N-H HSQC光谱中没有可检测到的信号。温度变化研究使我们能够确定这些快速交换酰胺质子的表观活化能在47-69 kJ mol范围内，这与氢氧化物催化的交换一致。
