Berger, Stefan

Brand, Torsten

Cabrita, Eurico J.

Hofmann, Hans-Jörg

Morris, Gareth A.

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 Residue Specific Studies of NH Exchange Rates Performed on Ubiquitin  Torsten Branda, Gareth A. Morrisb, Eurico J. Cabritac, Hans-Jörg Hofmannd,  Stefan Bergeraa Institute of Analytical Chemistry, University of Leipzig, Linnéstraße 3, 04103 Leipzig, Germany; E-Mail: tbrand@rz.uni-leipzig.de b Department of Chemistry, University of Manchester, Oxford Road, Manchester, M139PL, UK c Requimte/CQFB, Dep. de Quimica, Univ. Nova de Lisboa, 2829-516 Caparica, Portugal d Institute of Biochemistry, University of Leipzig, Brüderstraße 34, 04103 Leipzig, Germany 1. Introduction The aim of the present study was to establish a new method to investigate NH exchange rates in proteins. Experiments, which are based on residue specific diffusion measurements, were performed on human ubiquitin since it is a relatively small protein consisting of 76 residues, resulting in a molecular weight of 9 kDa. Furthermore, the availability of resonance assignment [1] as well as an NMR structure [2] is advantageous. 2. Experimental For NMR studies a 1.8 mM solution of uniformly 13C,15N-labelled ubiquitin in 90% H2O, 10% D2O, buffered to pH 5.8 was used. Experiments were carried out at 300 K on a Bruker Avance 700 spectrometer equipped with a cryo probe. Diffusion Ordered Spectroscopy [3] was used to obtain an apparent diffusion coefficient D for each separated signal. In order to observe resolved signals for most residues of the protein 1H,15N-DOSY-HSQC spectra were recorded. 1H,13C-DOSY-HSQC spectra were used as control experiments since CH protons cannot be affected by exchange. All separated CH signals of ubiquitin showed apparent diffusion coefficients which are in agreement with the actual size of the protein. Further control experiments using suppression of convection artefacts by double stimulated echo [4] were performed to prove that convection has no significant influence on apparent diffusion coefficients under the conditions used in this study. Due to sensitivity reasons, measurements for the determination of exchange rates were performed without suppression of convection artefacts.  3. Results Analysis of the apparent diffusion coefficients showed several “fast moving” NH protons. Subsequently, the exchange rates were derived directly from the decay data of the diffusion experiment by applying a model deduced from the assumption of two-site exchange with water and the “pure” diffusion coefficients of water and protein. © 2005, T. Brand The Open-Access Journal for the Basic Principles of Diffusion Theory, Experiment and ApplicationDiffusion Fundamentals 2 (2005) 66.1 - 66.2 1 The “pure” diffusion coefficient of the protein was determined in an experiment with selective excitation of the amide protons in order to prevent the influence of magnetization transfer from water to amide protons on the decay data. This value was in agreement with those diffusion coefficients obtained in 1H,13C-DOSY-HSQC experiments. Figure 1 shows the exchange rates obtained for ubiquitin.  NH exchange rates of ubiquitin0510152025300 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75residue indexk / s-1  We plan to compare our results to exchange rates determined by other NMR methods such as the MEXICO sequence [5]. These experiments are topic of current research.  4. Conclusion Diffusion measurements are a convenient tool for the detection of residues undergoing fast NH exchange and can complement relaxation studies. Exchange rates can also be quantitatively evaluated.  References [1] A. J. Wand, J. L. Urbauer, R. P. McEvoy, R. J. Bieber, Biochemistry 35 (1996) 6116-6125. [2] G. Cornilescu, J. L. Marquardt, M. Ottiger, A. Bax, J. Am. Chem. Soc. 120 (1998) 6836-6837.  [3] K. F. Morris, C. S. Johnson, J. Am. Chem. Soc. 114 (1992) 3139-3141. [4] A. Jerschow, N. Müller, J. Magn. Reson. 125 (1997) 372-375. [5] G. Gemmecker, W. Jahnke, H. Kessler, J. Am. Chem. Soc. 115 (1993) 11620-11621. Diffusion Fundamentals 2 (2005) 66.1 - 66.2 2

Residue specific studies of NH exchange rates performed on ubiquitin

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Residue specific studies of NH exchange rates performed on ubiquitin

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