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Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

ABSTRACT: Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron–electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to ‘knock out’ superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00723-021-01350-1.

SUBMITTER: Heaven G

PROVIDER: S-EPMC8550513 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeVelocity-selective arterial spin labeling (VSASL) has been proposed for renal perfusion imaging to mitigate planning challenges and effects of arterial transit time (ATT) uncertainties. In VSASL, label generation may shift in the vascular tree as a function of cutoff velocity. Here, we investigate label dynamics and especially the ATT of renal VSASL and compared it with a spatially selective pulsed arterial spin labeling technique, flow alternating inversion recovery (FAIR).MethodsArterial spin labeling data were acquired in 7 subjects, using free-breathing dual VSASL and FAIR with five postlabeling delays: 400, 800, 1200, 2000, and 2600 ms. The VSASL measurements were acquired with cutoff velocities of 5, 10, and 15 cm/s, with anterior-posterior velocity-encoding direction. Cortical perfusion-weighted signal, temporal SNR, quantified renal blood flow, and arterial transit time were reported.ResultsIn contrast to FAIR, renal VSASL already showed fairly high signal at the earliest postlabeling delays, for all cutoff velocities. The highest VSASL signal and temporal SNR was obtained with a cutoff velocity of 10 cm/s at postlabeling delay = 800 ms, which was earlier than for FAIR at 1200 ms. Fitted ATT on VSASL was ≤ 0 ms, indicating ATT insensitivity, which was shorter than for FAIR (189 ± 79 ms, P < .05). Finally, the average cortical renal blood flow measured with cutoff velocities of 5 cm/s (398 ± 84 mL/min/100 g) and 10 cm/s (472 ± 160 mL/min/100 g) were similar to renal blood flow measured with FAIR (441 ± 84 mL/min/100 g) (P > .05) with good correlations on subject level.ConclusionVelocity-selective arterial spin labeling in the kidney reduces ATT sensitivity compared with the recommended pulsed arterial spin labeling method, as well as if cutoff velocity is increased to reduce spurious labeling due to motion. Thus, VSASL has potential as a method for time-efficient, single-time-point, free-breathing renal perfusion measurements, despite lower tSNR than FAIR.

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Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

Supplementary Information

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