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

Abstract

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein–protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes’ local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin’s variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.

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

“…While all of the observed probe-induced thermodynamic perturbations in binding are small, the largest perturbations do NOT appear at sites structurally implicated as “most important” in the bound state. When considered along with similar recent measurements for labels placed on CaM, 62 the data in Table 2 strongly indicate that the C* probe residue can be placed directly along binding interfaces without strong perturbation of the binding.…”

“…However, some conclusions do emerge here for “appropriate use” of this probe group along binding interfaces. The ITC data and IR data together suggest that there is no major perturbation of the average bound structure by the probe placement at any of the selected sites (and a similar lack of perturbation was recently observed from the other side of the same interface 62 ), so assessing site-specific binding qualitatively through shifts in the mean or mode frequency should be possible in many systems, including those that are less structurally understood than the CaM-M13 complex. Despite its polarity, C* can apparently be placed in relatively hydrophobic environments without functional consequences.…”

“… 57 − 59 Each of the probes has a different solvent exposure in the bound complex (SASA estimates in Table 3 ), and solvation does not appear to be a major factor in the ΔΔ H and ΔΔ S values. Despite CaM’s highly conserved sequence, CaM binding has been generally shown (see below) to be somewhat mutation-tolerant, but our ITC data for both labels on the M13 peptide in this work and labels on CaM 62 show that C* is functionally nonperturbative to its environment in this particular complex.…”

Calmodulin and Calmodulin Binding Proteins in Dictyostelium: A Primer

“…CaM is the primary sensor and effector of calcium signal transduction in eukaryotes [8]. It functions in turn by binding to over 300 different CaM-binding proteins (CaMBPs) that mediate hundreds of cellular functions [9][10][11]. Despite this, most papers on calcium function and signal transduction fail to determine if CaM is at play.…”

“…Because of the dynamic and extensive conformational events induced by first calcium binding and then CaMBP binding, CaM has been and still is actively studied by chemists, biochemists, structural, and theoretical biologists to name a few. New insights into these events continue to be revealed using new technologies (e.g., [10,23,24] Here, we will examine the fundamentals of these binding events with a view to their impact on the structure and function of Dictyostelium CalA.…”