Population Dynamics of Stretching Excitations of p-Azido-phenylalanine Incorporated in Calmodulin-Peptide Complexes.

We genetically incorporated the unnatural amino acid p-azido-phenylalanine (AzF) into the ubiquitous Ca2+ sensor protein calmodulin (CaM) in complex with different peptides to explore the response of the azido stretching line shape to varying binding motifs with femtosecond infrared spectroscopy. The dynamic response of the azido stretching mode varies in different CaM-peptide complexes. We model these dynamics as coherent excitations of Fermi resonances and extract a lifetime of the azido stretching vibration of about 1 ps. The resulting model parameters are commensurate with the linear infrared absorption lineshapes which suggests that the conformation-sensitive vibrational lineshape could be composed of Fermi resonances that differ between the protein-peptide complexes.

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

Calmodulin (CaM) is a very conserved, ubiquitous, eukaryotic protein that binds four Ca2+ ions with high affinity. It acts as a calcium sensor by translating Ca2+ signals into cellular processes such as metabolism, inflammation, immune response, memory, and muscle contraction. Calcium binding to CaM leads to conformational changes that enable Ca2+/CaM to recognize and bind various target proteins with high affinity. The binding mode and binding partners of CaM are very diverse, and a consensus binding sequence is lacking. Here, we describe an elegant system that allows conformation-specific detection of CaM-binding to its binding partners. We incorporate the unnatural amino acid p-azido-phenylalanine (AzF) in different positions of CaM and follow its unique spectral signature by infrared (IR)-spectroscopy of the azido stretching vibration. Our results suggest that the AzF vibrational probe is sensitive to the chemical environment in different CaM/CaM-binding domain (CaMBD) complexes, which allows differentiating between different binding motifs according to the spectral characteristics of the azido stretching mode. We corroborate our results with a crystal structure of AzF-labelled CaM (CaM108AzF) in complex with a binding peptide from calmodulin-dependent protein kinase IIα identifying the structural basis for the observed IR frequency shifts.

Reformulation of the D3(Becke-Johnson) Dispersion Correction without Resorting to Higher than C6 Dispersion Coefficients.

A reformulated version of Grimme's most recent DFT dispersion correction with Becke-Johnson damping (DFT-D3(BJ)) is presented, which only depends on C6 dispersion coefficients. The role of the higher order correction terms in the DFT-D3(BJ) model is critically investigated, and a sigmoidal interpolation function for adjusting to different density functional approximations (DFA) is employed alternatively, while keeping finite damping of Becke and Johnson. For the proposed C6-only dispersion correction scheme, only one parameter needs to be fitted per DFA (instead of three for DFT-D3(BJ)). Eight standard DFAs from different classes are parametrized and evaluated. In comparison to DFT-D3(BJ), one of the most accurate corrections up to date, the new correction shows only negligible deviations in accuracy for the huge GMTKN30 benchmark set.