Validating the 15N-1H HSQC-ROESY experiment for detecting 1HN exchange broadening in proteated proteins

• Published in: Journal of magnetic resonance (1997) Vol. 363; p. 107676
• Main Author: Zuiderweg, Erik R.P.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.06.2024
• Subjects: Amide proton relaxation; Proteins; R1rho; Relaxation dispersion; Solution NMR; Proteins; R1rho; Amide proton relaxation; Relaxation dispersion; Solution NMR
• ISSN: 1090-7807; 1096-0856; 1096-0856
• DOI: 10.1016/j.jmr.2024.107676

[Display omitted] •Milli-to-micro-second conformational exchange data contained in 1HN NMR R2 protein relaxation data is reinvestigated.•The 1HN relaxation terms for a fully proteated protein are theoretically analyzed.•Multi-spin cross correlations are quantitated and exploited.•TOCSY coherence transfer is quantified and eliminated.•ROESY cross peaks are eliminated. It is advantageous to investigate milli-to-micro-second conformational exchange data contained in the solution NMR protein relaxation data other than 15N nuclei. Not only does one search under another lamp post, one also looks at dynamics at other time scales. The HSQC-ROESY 1HN relaxation dispersion experiment for amide protons as introduced by Ishima, et al (1998). J. Am. Soc. 120, 10534–10542, is such an experiment, but has by the authors been advised to only be used for perdeuterated proteins to avoid complication with the 1H–1H multiple-spin effects. This is regretful, since not all proteins can be perdeuterated. Here we analyze in detail the 1HN relaxation terms for this experiment for a fully proteated protein. Indeed, the 1HN relaxation theory is in this case complex and includes dipolar-dipolar relaxation interference and TOCSY transfers. With simulate both of these effects and show that the interference can be exploited for detecting exchange broadening. The TOCSY effect is shown to minor, and when it is not, a solution is provided. We apply the HSQC-ROESY experiment, with a small modification to suppress ROESY crosspeaks, to a 7 kDa GB1 protein that is just 15N and 13C labeled. At 10 °C we cannot detect any conformational exchange broadening: the 1HN R2 relaxation rates with 1.357 kHz spinlock field not larger than those recorded with a 12.136 kHz spinlock field. This means that there is no exchange broadening that can be differentially suppressed with the applied fields. Either there is no broadening, or the broadening is effectively suppressed by all fields, or the broadening cannot be suppressed by either of the fields. While initially this seems to be a disappointing result, we feel that this work establishes that the HSQC-ROESY experiment is very robust. It can indeed be utilized for proteated proteins upto about 30 kDa. This could be opening the study the milli-microsecond conformational dynamics as reported by 1HN exchange broadening for many more proteins.