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

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.