Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: Substrate Binding Switches the Conformation at the Lynchpin Site in the Substrate-Binding Domain of Human Hsp70 to Enable Allosteric Interdomain Communication

doi: 10.3390/molecules23030528

Figure Lengend Snippet: PRE experiments with SBD(∆CDE)-y with spin labeling at residue 537. ( a ) The spin-labeling position in the solution structure of SBD(∆CDE)-y is shown, in which, S537 was exchanged to cysteine to facilitate covalent labeling with MTSL; ( b ) Representative spectral changes caused by the paramagnetic effect. In the left two panels, 2D 1 H- 15 N HSQC spectra are overlaid for the paramagnetic spin labeled sample (red) and for the sample with a diamagnetic label (black) in the absence of the NR-peptide. In the two panels on the right, the corresponding spectral comparison in the presence of 5.0 mM NR-peptide. The concentration of SBD(∆CDE)-y was adjusted to 0.5 mM.

Article Snippet: The SBD(∆CDE) (S537C/L542Y) mutant was incubated with 10-fold molar excess of (1-oxyl-2,2,5,5-tetramethyl-delta(3)-pyrroline-3-methyl)-methanethiosulfonate (MTSL), or a diamagnetic control MTSL analog, (1-acetoxy-2,2,5,5-tetramethyl-δ-3-pyrroline-3-methyl)methane-thiosulfonate (Toronto Research Chemicals, Toronto, ON, Canada), dissolved in DMSO at 296 K (23 °C) for 10 h. The sample solution was dialyzed extensively against buffer E at 277 K (4 °C) to remove unreacted MTSL.

Techniques: Labeling, Residue, Comparison, Concentration Assay