Self-assembling multidomain peptides have been shown to have desirable properties, such as the ability to form hydrogels that rapidly recover following shear-thinning and the potential to be tailored by amino acid selection to vary their elasticity and encapsulate and deliver proteins and cells. Here we describe the effects of substitution of aliphatic hydrophobic amino acids in the central domain of the peptide for the aromatic amino acids phenylalanine, tyrosine and tryptophan. While the basic nanofibrous morphology is retained in all cases, selection of the particular core residues results in switching from anti-parallel hydrogen bonding to parallel hydrogen bonding in addition to changes in nanofiber morphology and in hydrogel rheological properties. Peptide nanofiber assemblies are investigated by circular dichroism polarimetry, infrared spectroscopy, atomic force microscopy, transmission and scanning electron microscopy, oscillatory rheology and molecular dynamics simulations. Results from this study will aid in designing next generation cell scaffolding materials.

Hsp70 molecular chaperones play an important role in maintaining cellular homeostasis, and are implicated in a wide array of cellular processes, including protein recovery from aggregates, cross-membrane protein translocation, and protein biogenesis. Hsp70 consists of two domains, a nucleotide binding domain (NBD) and a substrate binding domain (SBD), each of which communicates via an allosteric mechanism such that the protein interconverts between two functional states, an ATP-bound open conformation and an ADP-bound closed conformation. The exact mechanism for interstate conversion is not as yet fully understood. However, the ligand-bound states of the NBD and SBD as well as interactions with cochaperones such as DnaJ and nucleotide exchange factor are thought to play crucial regulatory roles. In this study, we apply the perturbation-response scanning (PRS) method in combination with molecular dynamics simulations as a computational tool for the identification of allosteric hot residues in the large multidomain Hsp70 protein. We find evidence in support of the hypothesis that substrate binding triggers ATP hydrolysis and that the ADP-substrate complex favors interstate conversion to the closed state. Furthermore, our data are in agreement with the proposal that there is an allosterically active intermediate state between the open and closed states and vice versa, as we find evidence that ATP binding to the closed structure and peptide binding to the open structure allosterically "activate" the respective complexes. We conclude our analysis by showing how our PRS data fit the current opinion on the Hsp70 conformational cycle and present several allosteric hot residues that may provide a platform for further studies to gain additional insight into Hsp70 allostery.

Conformational transitions in proteins facilitate precise physiological functions. Therefore, it is crucial to understand the mechanisms underlying these processes to modulate protein function. Yet, studying structural and dynamical properties of proteins is notoriously challenging due to the complexity of the underlying potential energy surfaces (PES). We have previously developed the perturbation-response scanning (PRS) method to identify key residues that participate in the communication network responsible for specific conformational transitions. PRS is based on a residue-by-residue scan of the protein to determine the subset of residues/forces which provide the closest conformational change leading to a target conformational state, inasmuch as linear response theory applies to these motions. Here, we develop a novel method to further evaluate if conformational transitions may be triggered on the PES. We aim to study functionally relevant conformational transitions in proteins by using results obtained from PRS and feeding them as inputs to steered molecular dynamics simulations. The success and the transferability of the method are evaluated on three protein systems having different complexities of motion on the PES: calmodulin, adenylate kinase, and bacterial ferric binding protein. We find that the method captures the target conformation, while providing key residues and the optimum paths with relatively low free energy profiles.

Helix-8 (Hx8) is a structurally conserved amphipathic helical motif in class-A GPCRs, adjacent to the C-terminal sequence that is responsible for PDZ-domain-recognition. The Hx8 segment in the dopamine D2 receptor (D2R) constitutes the C-terminal segment and we investigate its role in the function of D2R by studying the interaction with the PDZ-containing GIPC1 using homology models based on the X-ray structures of very closely related analogs: the D3R for the D2R model, and the PDZ domain of GIPC2 for GIPC1-PDZ. The mechanism of this interaction was investigated with all-atom unbiased molecular dynamics (MD) simulations that reveal the role of the membrane in maintaining the helical fold of Hx8, and with biased MD simulations to elucidate the energy drive for the interaction with the GIPC1-PDZ. We found that it becomes more favorable energetically for Hx8 to adopt the extended conformation observed in all PDZ-ligand complexes when it moves away from the membrane, and that C-terminus palmitoylation of D2R enhanced membrane penetration by the Hx8 backbone. De-palmitoylation enables Hx8 to move out into the aqueous environment for interaction with the PDZ domain. All-atom unbiased MD simulations of the full D2R-GIPC1 complex in sphingolipid/cholesterol membranes shows that the D2R carboxyl C-terminus samples the region of the conserved GFGL motif located on the carboxylate-binding loop of the GIPC1-PDZ, and the entire complex distances itself from the membrane interface. Together, these results outline a likely mechanism of Hx8 involvement in the interaction of the GPCR with PDZ-domains in the course of signaling.

One of the major challenges in the development of coarse grained (CG) simulation models that aim at biomolecular structure formation processes is the correct representation of an environment-driven conformational change, for example, a folding/unfolding event upon interaction with an interface or upon aggregation. In the present study, we investigate this transferability challenge for a CG model using the example of diphenylalanine. This dipeptide displays a transition from a trans-like to a cis-like conformation upon aggregation as well as upon transfer from bulk water to the cyclohexane/water interface. Here, we show that one can construct a single CG model that can reproduce both the bulk and interface conformational behavior and the segregation between hydrophobic/hydrophilic medium. While the general strategy to obtain nonbonded interactions in the present CG model is to reproduce solvation free energies of small molecules representing the CG beads in the respective solvents, the success of the model strongly depends on nontrivial decisions one has to make to capture the delicate balance between the bonded and nonbonded interactions. In particular, we found that the peptide's conformational behavior is qualitatively affected by the cyclohexane/water interaction potential, an interaction that does not directly involve the peptide at all but merely influences the properties of the hydrophobic/hydrophilic interface. Furthermore, we show that a small modification to improve the structural/conformational properties of the CG model could dramatically alter the thermodynamic properties.