Buffer effects on site directed Cu2+-labeling using the double histidine motif.

The double histidine, or dHis, motif has emerged as a powerful spin labeling tool to determine the conformations and dynamics, subunit orientation, native metal binding site location, and other physical characteristics of proteins by Cu2+-based electron paramagnetic resonance. Here, we investigate the efficacy of this technique in five common buffer systems, and show that buffer choice can impact the loading of Cu2+-NTA into dHis sites, and more generally, the sensitivity of the overall technique. We also present a standardized and optimized examination of labeling of the dHis motif with Cu2+-NTA for EPR based distance measurements. We provide optimal loading procedures, using representative EPR and UV/Vis data for each step in the process. From this data, we find that maximal dHis loading can be achieved in under 30 min with low temperature sample incubation. Using only these optimal procedures, we see up to a 28% increase in fully labeled proteins compared to previously published results in N-ethylmorpholine. Using both this optimized procedure as well as a more optimal buffer, we can achieve up to 80% fully loaded proteins, which corresponds to a 64% increase compared to the prior data. These results provide insight and deeper understanding of the dHis Cu2+-NTA system, the variables that impact its efficacy, and present a method by which these issues may be mitigated for the most efficient labeling.