bi8b00283_si_001.pdf (1.06 MB)
Cyanylated Cysteine Reports Site-Specific Changes at Protein–Protein-Binding Interfaces Without Perturbation
journal contribution
posted on 2018-05-22, 00:00 authored by Shannon
R. Dalton, Alice R. Vienneau, Shana R. Burstein, Rosalind J. Xu, Sara Linse, Casey H. LonderganTo investigate the
cyanylated cysteine vibrational probe group’s
ability to report on binding-induced changes along a protein–protein
interface, the probe group was incorporated at several sites in a
peptide of the calmodulin (CaM)-binding domain of skeletal muscle
myosin light chain kinase. Isothermal titration calorimetry was used
to determine the binding thermodynamics between calmodulin and each
peptide. For all probe positions, the binding affinity was nearly
identical to that of the unlabeled peptide. The CN stretching infrared
band was collected for each peptide free in solution and bound to
calmodulin. Binding-induced shifts in the IR spectral frequencies
were correlated with estimated solvent accessibility based on molecular
dynamics simulations. This work generally suggests (1) that site-specific
incorporation of this vibrational probe group does not cause major
perturbations to its local structural environment and (2) that this
small probe group might be used quite broadly to map dynamic protein-binding
interfaces. However, site-specific perturbations due to artificial
labeling groups can be somewhat unpredictable and should be evaluated
on a site-by-site basis through complementary measurements. A fully
quantitative, simulation-based interpretation of the rich probe IR
spectra is still needed but appears to be possible given recent advances
in simulation techniques.