Supplementary Figure 3: Spin-labeling efficiency on KvAP-VSD cysteine mutant.

(a) ESI-TOF mass spectrometry analysis for labeled (with either MTSL or bifunctional spin label) KvAP VSD cysteine mutants (either 118C/121C or 121C). The m/z of ionized fragments (top) and deconvoluted mass (bottom) were shown for each of four samples. The spin labeled protein has predominant peak in the MS spectra. (b) Summary of the MS results confirming the correct bi-functional attachment of Bi-SL onto the sensor. In particular, there are three possibilities for a bifunctional spin label attaching to double cysteine mutant: 1. Sinlge Bi-SL on one of the cysteine (expected MW, 16954.7); 2. Two Bi-SL on both single cysteine (expected MW, 17262.8); 3. Single Bi-SL on two cysteine (expected MW, 16874.5). The MS unambiguously confirms that both reaction group of Bi-SL were removed through reaction with two cysteine groups. (c) Continuous EPR spectra of MTSL labeled (left) and Bi-SL labeled (right) samples. Two MTSL on to adjacent positions shows characteristic dipolar coupling (left, red). At the labeling condition with 20X molar excess of spin label, if a Bi-SL didn't react with both cysteines together, the double cysteine mutant would be labeled with two Bi-SL which will show significant bipolar coupling feature in CW-EPR spectra.