Elsevier

Journal of Magnetic Resonance

Volume 249, December 2014, Pages 32-37
Journal of Magnetic Resonance

H/D exchange of a 15N labelled Tau fragment as measured by a simple Relax-EXSY experiment

https://doi.org/10.1016/j.jmr.2014.10.008Get rights and content

Highlights

  • We propose a novel H/D exchange experiment to measure amide exchange rates.

  • The method requires only 15N labelling and can be used in intrinsically unfolded proteins.

  • Amide exchange rates in a functional fragment of Tau are measured.

  • The rates in a phosphorylated fragment demonstrate an intraresidue hydrogen bond.

Abstract

We present an equilibrium H/D exchange experiment to measure the exchange rates of labile amide protons in intrinsically unfolded proteins. By measuring the contribution of the H/D exchange to the apparent T1 relaxation rates in solvents of different D2O content, we can easily derive the rates of exchange for rapidly exchanging amide protons. The method does not require double isotope labelling, is sensitive, and requires limited fitting of the data. We demonstrate it on a functional fragment of Tau, and provide evidence for the hydrogen bond formation of the phosphate moiety of Ser214 with its own amide proton in the same fragment phosphorylated by the PKA kinase.

Introduction

Amide hydrogen exchange is a powerful tool to obtain information about structure and structural dynamics of proteins and nucleic acids [1]. First introduced by Linderstrøm-Lang [2], [3], [4], [5], it has proven a valuable tool for protein studies including folding and refolding pathways [6], conformational changes [7], dynamics [8], structure determination [9], [10], [11] and protein–protein interactions [12], [13]. Several methods are capable of measuring accurately the protein solvent exchange: mass spectroscopy [12], [14], [15], neutron crystallography [16], [17], and NMR [18], [19], [20]. Although NMR is limited by the size of the protein and by the requirement of isotope labelling, it remains a privileged technique to measure exchange rates as it allows a resolution at the level of an individual amino acid with minimal interference with the sample, and data measurement is straightforward. Rapid dissolving of a protonated protein sample into a deuterated buffer and subsequent monitoring of decreasing amide proton intensities thereby is the simplest experiment, but it is limited to the cases of slow H/D exchange [18]. Manipulating the water magnetization and observing how this affects the amide proton intensity is an alternative approach in the case of rapid H/D exchange [21], [22], [23], [24], [25], [26]. However, this approach suffers from a number of experimental drawbacks, with radiation damping being a non negligible problem on the high magnetic fields presently available. Moreover, as the water line coincides with a number of Hα protons, magnetization transfer by a dipolar NOE transfer from the Hα to the amide protons can be misinterpreted as chemical exchange with the water magnetization. The CLEANEX-PM [26] method was developed to eliminate this unwanted NOE transfer in the limit of high τc values, by compensating the possible NOE transfer by a rotating frame ROE transfer of opposite sign [27].

Recently, the H/D-SOLEXSY [28] was proposed to measure fast proton deuterium exchange at equilibrium in a mixed H2O/D2O buffer as solvent. The mixed solvent thereby assures the presence of two distinct populations of amide nitrogens, bound to either a proton or a deuteron, that can be distinguished through the differential isotope effect of the 1H or 2D nucleus on the 15N chemical shift value. Detection of the two nitrogen frequencies starting from the Hα proton of the previous residue (based on the HA(CACO)NH pulse sequence) allows the direct measurement of the exchange between both cross peaks. Although capable of measuring fast water exchange rates in folded and unfolded proteins, this approach requires 15N/13C labelling, is limited in sensitivity because of multiple magnetization transfer steps involving comparatively faster relaxation rates of Hα and Cα, and requires complex data analysis by fitting several unknown parameters.

The influence of the H/D exchange on the apparent longitudinal relaxation rate has been described [29], [30], and was found to be a potential source of error when exchange is rapid. Here, we propose to exploit it in a constructive manner to extract the exchange rates, and make it more robust by multiplying the T1 measurements of a given sample in aqueous buffers with variable percentages of H2O and D2O. The Relax-EXSY (Relaxation based EXchange SpectroscopY) experiment is thereby a simpler version of H/D-SOLEXSY, and the contribution of the chemical H/D exchange to the apparent T1 rate can readily be extracted for each residue. The Relax-EXSY experiment does not require 13C labelling, and avoids any lengthy 15N to 13C coherence transfer, thereby gaining considerable sensitivity over the H/D-SOLEXSY experiment. Because of this increased sensitivity, the measurement can easily be performed with several H2O/D2O ratios to increase its accuracy. The experiment does not rely upon the manipulation of the water magnetization, and the data can be fitted with only two unknown parameters.

H/D exchange has recently been applied to the study of intrinsically unfolded proteins [31], and we first illustrate the Relax-EXSY method by measuring the amide exchange rates for the different amino acids of the TauF4 fragment. This fragment, that spans residues [208–334] from the neuronal Tau protein, is fully functional in terms of microtubule assembly [32] but also forms the Alzheimer’s disease characteristic fibres when incubated with heparin [33]. We validate the method by measuring the exchange rates of the same sample with the H/D-SOLEXSY method, and equally use theoretical values parameterized on the basis of exchange rates measured on model peptides [34] to see whether the resulting exchange rates reveal any residual structure in this Tau fragment. We finally apply the Relax-EXSY method to explore the amide proton protection due to phosphorylation of a Serine in the same TauF4 fragment.

Section snippets

Results and discussion

As in the 15N T1 relaxation experiment, amide proton magnetisation is transferred during the first refocused INEPT transfer period (Fig. 1, time points a to b) to its nitrogen, resulting in a – NHz magnetization term for the protonated amide groups. Deuterated amide nitrogen atoms will not lead to detectable magnetization, as the corresponding term will be destroyed by the G2 pulse field gradient. During the mixing time (points b to c, Fig. 1), the – NHz term will return to its equilibrium

Conclusion

We have presented here a simple and highly sensitive experiment to measure the H/D exchange rates for amide protons in an intrinsically unfolded protein. Its high sensitivity, easy implementation and straightforward fitting of the desired parameters should make it into a method of choice for measuring the amide exchange rates of rapidly exchanging amide protons by NMR. We have used the method to probe amide exchange in a functional fragment of Tau, and to show that the strong hydrogen bond

Experimental

The TauF4 fragment was produced and purified as previously described [32]. Phosphorylation of TauF4 was accomplished by in vitro phosphorylation of the recombinant sample by the PKA kinase as described previously [50]. Limiting the incubation time of the sample with the recombinant kinase to 10 min ensures the single but complete phosphorylation of the Ser214 residue in TauF4.

All the experiments were performed at 25 °C on Bruker Avance I spectrometer operating at 600 MHz proton frequency, and

Acknowledgments

We thank Prof. Skrynnikov for providing the SOLEXSY NMR sequence. The research leading to these results was supported by the Centre National de la Recherche Scientifique, the Agence Nationale de la Recherche (ANR-05-Blanc-6320-01, and program MALZ-TAF), the LABEX (laboratory of excellence program investment for the future) DISTALZ grant (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer’s disease), and the CNRS Large Scale Facility NMR THC Fr3050. The NMR

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