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CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions

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Abstract

1H–15N HSQC spectroscopy is a workhorse of protein NMR. However, under physiological conditions the quality of HSQC spectra tends to deteriorate due to fast solvent exchange. For globular proteins only a limited number of surface residues are affected, but in the case of intrinsically disordered proteins (IDPs) HSQC spectra are thoroughly degraded, suffering from both peak broadening and loss of intensity. To alleviate this problem, we make use of the following two concepts. (1) Proton-decoupled HSQC. Regular HSQC and its many variants record the evolution of multi-spin modes, 2NxHz or 2NxHx, in indirect dimension. Under the effect of fast solvent exchange these modes undergo rapid decay, which results in severe line-broadening. In contrast, proton-decoupled HSQC relies on Nx coherence which is essentially insensitive to the effects of solvent exchange. Moreover, for measurements involving IDPs at or near physiological temperature, Nx mode offers excellent relaxation properties, leading to very sharp resonances. (2) Cross-polarization 1H-to-15N transfer. If CP element is designed such as to lock both 1HN and water magnetization, the following transfer is effected: \( {\text{H}}_{\text{x}}^{\text{water}} \to {\text{H}}_{\text{x}}^{\text{N}} \to {\text{N}}_{\text{x}} . \) Thus water magnetization is successfully exploited to boost the amount of signal. In addition, CP element suffers less loss from solvent exchange, conformational exchange, and dipolar relaxation compared to the more popular INEPT element. Combining these two concepts, we have implemented the experiment termed CP-HISQC (cross-polarization assisted heteronuclear in-phase single-quantum correlation). The pulse sequence has been designed such as to preserve water magnetization and therefore can be executed with reasonably short recycling delays. In the presence of fast solvent exchange, kex ~ 100 s−1, CP-HISQC offers much better spectral resolution than conventional HSQC-type experiments. At the same time it offers up to twofold gain in sensitivity compared to plain proton-decoupled HSQC. The new sequence has been tested on the sample of drkN SH3 domain at pH 7.5, 30 °C. High-quality spectrum has been recorded in less than 1 h, containing resonances from both folded and unfolded species. High-quality spectra have also been obtained for arginine side-chain HεNε groups in the sample of short peptide Sos. For Arg side chains, we have additionally implemented (HE)NE(CD)HD experiment. Using 13C-labeled sample of Sos, we have demonstrated that proton-to-nitrogen CP transfer remains highly efficient in the presence of solvent exchange as fast as kex = 620 s−1. In contrast, INEPT transfer completely fails in this regime.

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Notes

  1. The alternative sample conditions could be pH 7.2, 37 °C. These conditions are relevant for cytosol (Orij et al. 2009). They give rise to very similar kex rates and are also suitable for CP-HISQC measurements.

  2. In principle, loss of multispin correlations due to solvent exchange can also be described as "relaxation". However, we prefer to reserve this term for faster processes—specifically for the dynamic regime where JNH/kex ≤ 1 (including Redfield limit where JNH/kex « 1 and the formula for scalar relaxation of the first kind applies (Abragam 1961)). Since in our study these conditions are not necessarily fulfilled we prefer a more general description referring to the "loss of multispin correlations due to solvent exchange" (Skrynnikov and Ernst 1999). In what follows the term "relaxation" refers to dipolar and CSA mechanisms.

  3. This general discussion is also valid in the situation when INEPT element includes pulsed field gradients.

  4. This parameter is the same as \( {\text {tan}} \, \uptheta \), where θ is the standard tilt angle.

  5. With this new hardware it is likely possible to execute the CP-HISQC experiment with shorter recycling delays. If in addition one can speed up the recovery of water magnetization, e.g. by introducing paramagnetic water relaxation agents (Hiller et al. 2005; Theillet et al. 2011), this can further improve the sensitivity of the CP-HISQC experiment. One should be mindful, however, that relaxation agents may perturb a delicate conformational equilibrium in the sample of an IDP.

  6. Note that in the case of (fully or partially) folded proteins the saturation of the water signal does not only hurt labile 1HN protons, but also those protons that are protected from solvent exchange. Specifically, the saturation is transferred via spin diffusion from solvent-exposed protons on the surface of the protein to protons in the hydrophobic core, thus reducing the amount of magnetization across the board.

  7. Of interest, certain advanced schemes exist to align water magnetization along the effective rf field (Hansen and Kay 2007). These schemes, however, are only useful for spin-lock experiment and do not help in the case of phase-alternated pulse trains such as DIPSI-2.

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Acknowledgments

This study was supported by the funds from NSF Grant MCB 1158347.

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  1. Department of Chemistry, Purdue University, West Lafayette, IN, 47907, USA

    Tairan Yuwen & Nikolai R. Skrynnikov

  2. Laboratory of Biomolecular NMR, St. Petersburg State University, St. Petersburg, 199034, Russia

    Nikolai R. Skrynnikov

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  1. Tairan Yuwen
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Correspondence to Nikolai R. Skrynnikov.

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Yuwen, T., Skrynnikov, N.R. CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions. J Biomol NMR 58, 175–192 (2014). https://doi.org/10.1007/s10858-014-9815-5

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