We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM–DAPK2 S308D mutant peptide is also reported. The structures of CaM–DAPK peptide complexes present a mode of CaM–kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the largest contribution coming from the Trp305. About two-thirds of the buried surface area corresponds to nonpolar residues, showing that hydrophobic interactions play an important role in these CaM–peptide complexes. The simulation results agree with the experimental data in predicting a small effect of the S308D mutation on CaM interactions with DAPK2, suggesting that this mutation is not a good model for the S308 phosphorylation.

We present a computer simulation study of helix folding in alanine homopeptides (ALA)n of length n = 5, 8, 15, and 21 residues. Based on multi-microsecond molecular dynamics simulations at room temperature, we found helix populations and relaxation times increasing from about 6% and ~2 ns for ALA5 to about 60% and ~500 ns for ALA21, and folding free energies decreasing linearly with the increasing number of residues. The helix folding was analyzed with the Optimal Dimensionality Reduction method, yielding coarse-grained kinetic models that provided a detailed representation of the folding process. The shorter peptides, ALA5 and ALA8, tended to convert directly from coil to helix, while ALA15 and ALA21 traveled through several intermediates. Coarse-grained aggregate states representing the helix, coil, and intermediates were heterogeneous, encompassing multiple peptide conformations. The folding involved multiple pathways and interesting intermediate states were present on the folding paths, with partially formed helices, turns, and compact coils. Statistically, helix initiation was favored at both termini, and the helix was most stable in the central region. Importantly, we found the presence of underlying universal local dynamics in helical peptides with correlated transitions for neighboring hydrogen bonds. Overall, the structural and dynamical parameters extracted from the trajectories are in good agreement with experimental observables, providing microscopic insights into the complex helix folding kinetics.

We present the results of microsecond length simulations of folding for three model helix-forming peptides, penta-alanine Ac-A₅-NH₂ (acA5a), a modified pentapeptide Ac-WA₃H⁺-NH₂ (WH5) and a 21-residue peptide Ac-WA₃H⁺-(AAARA)₃-A-NH₂ (WH21), employing three protein force fields, AMBER03, CHARMM27 and OPLS-AA. The calculated helix populations are in good agreement with observations for all peptides, and the measured kinetic rate constants of WH5 are well reproduced. Our results yield new and interesting insights into the mechanism of α-helix folding. For acA5a, representing a flexible generic helical nucleus, we find significant variation of the folding pathway with employed force field. For the WH5 helix, which includes a stabilising side chain interaction, the nucleation step also varies with force field, but an intermediate with first two hydrogen bonds formed is a common feature in all models. For WH21 the three employed force fields differed in details of the helix initiation stage. However, the main stages of folding, involving first the central region, then the N-terminal and finally the C-terminal, were very similar in AMBER03, CHARMM27 and OPLS-AA. Single-helix structures were dominant intermediates, with a minor contribution from helix-turn-helix conformers. The agreement between simulations and experimental data indicate that current protein force fields provide a reliable description α-helix folding.