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Simultaneous recording of intra- and inter-residue linking experiments for backbone assignments in proteins at MAS frequencies higher than 60 kHz
Journal of Biomolecular NMR ( IF 2.319 ) Pub Date : 2020-01-01 , DOI: 10.1007/s10858-019-00292-y
Kshama Sharma, P. K. Madhu, Vipin Agarwal, Kaustubh R. Mote

Obtaining site-specific assignments for the NMR spectra of proteins in the solid state is a significant bottleneck in deciphering their biophysics. This is primarily due to the time-intensive nature of the experiments. Additionally, the low resolution in the \(^{1}{\text {H}}\)-dimension requires multiple complementary experiments to be recorded to lift degeneracies in assignments. We present here an approach, gleaned from the techniques used in multiple-acquisition experiments, which allows the recording of forward and backward residue-linking experiments in a single experimental block. Spectra from six additional pathways are also recovered from the same experimental block, without increasing the probe duty cycle. These experiments give intra- and inter residue connectivities for the backbone \(^{13}{\text {C}}_\alpha\), \(^{15}{\text {N}}\), \(^{1}{\text {H}}_{\text {N}}\) and \(^{1}{\text {H}}_\alpha\) resonances and should alone be sufficient to assign these nuclei in proteins at MAS frequencies > 60 kHz. The validity of this approach is tested with experiments on a standard tripeptide N-formyl methionyl-leucine-phenylalanine (f-MLF) at a MAS frequency of 62.5 kHz, which is also used as a test-case for determining the sensitivity of each of the experiments. We expect this approach to have an immediate impact on the way assignments are obtained at MAS frequencies \(> 60\text { kHz}\).
更新日期:2020-01-01

 

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