Cyanylated Cysteine Reports Site-Specific Changes at Protein–Protein-Binding Interfaces Without Perturbation

Abstract

To 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.

Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups

“… 30 − 32 Replacement of native residues along a binding interface by β-thiocyano-alanine (or cyanylated cysteine, C* for short) is surprisingly nonperturbative both to the protein’s local secondary structure 33 and to protein–protein binding thermodynamics. 25 …”

“…We recently reported 25 dramatic changes in the CN stretching IR absorption bands of SCN labels at six different sites on the M13 peptide as CaM wrapped around it. Furthermore, we showed that the artificial C* residues on the target peptide did not substantially perturb the binding between the species.…”

Conformation-specific detection of calmodulin binding using the unnatural amino acid p-azido-phenylalanine (AzF) as an IR-sensor

“…Recently, binding of calmodulin to peptides has been investigated by IR spectroscopy using an unnatural amino acid (UAA) that was chemically introduced via cyanylated cysteine (-SCN) reporter labels on either CaM or binding peptides. 12,13 …”

Visualizing the movement of the amphipathic helix in the respiratory complex I using a nitrile infrared probe and SEIRAS

“…The thiocyanate moiety is generated by chemical modification of the free thiol of an accessible cysteine residue and can thus be inserted at any cysteine site either being a natural residue or resulting from mutagenesis . Previous studies on the influence of the microenvironment on the signature of the SC≡N stretching frequency in proteins showed typical signals at about 2160 cm −1 for labeled ribonuclease S and for the protein calmodulin .…”