Abstract
The signaling protein calmodulin (CaM) undergoes a well-known change in secondary structure upon binding Ca2+, but the structural plasticity of the Ca2+-free apo state is linked to CaM functionality. Variable temperature studies of apo-CaM indicate two structural transitions at 46 and 58 °C that are assigned to melting of the C- and N-terminal domains, respectively, but the molecular mechanism of domain unfolding is unknown. We report temperature-jump time-resolved infrared (IR) spectroscopy experiments designed to target the first steps in the C-terminal domain melting transition of human apo-CaM. A comparison of the nonequilibrium relaxation of apo-CaM with the more thermodynamically stable holo-CaM, with 4 equiv of Ca2+ bound, shows that domain melting of apo-CaM begins on microsecond time scales with α-helix destabilization. These observations enable the assignment of previously reported dynamics of CaM on hundreds of microsecond time scales to thermally activated melting, producing a complete mechanism for thermal unfolding of CaM.
Highlights
The link between structure and function in biological molecules is well-established and raises important questions given that proteins are dynamic in the solution phase
We report the use of high pulse repetition rate T-jump pumpIR probe spectroscopy[25] to target the fastest steps in the unfolding of the human calmodulin protein (CaM)
In the case of holo-CaM (Figure 1f), a loss of intensity near 1635 cm−1 was accompanied by a broad and rather featureless gain in intensity peaking near 1658 cm−1 but extending toward 1700 cm−1. These results have been assigned previously, with the aid of circular dichroism, differential scanning calorimetry (DSC), and 2D-IR spectroscopy, to the effects of increased temperature of the solvent combined, in the case of apo-CaM, with a helix-tocoil transition consistent with C-terminal domain melting at 46 °C.54
Summary
The link between structure and function in biological molecules is well-established and raises important questions given that proteins are dynamic in the solution phase This means that having the ability to follow the mechanisms of structural change in real time is imperative if we are to understand and modify protein behavior in vivo. T-jump methods have been used to access time scales ranging from the nanosecond pulse duration of the excitation laser to the milliseconds required for the temperature-jump to dissipate.[1−4,6−16] An alternative strategy employed solvated dyes to achieve a rapid temperature change,[17] while jumps in pH have been used to study peptide structural transitions.[18] An advantage in using temperature or pH to perturb proteins is the ability to explore their potential energy surface in the absence of structural modifications or non-natural entities.[19−22]. With a focus on time scales shorter than 100 μs, our results develop upon the single previous time-resolved study of CaM, which reported two-state unfolding of the C-terminal domain on several hundred microsecond time scales.[35]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
