The Role of the Interdomain Interactions on RfaH Dynamics and Conformational Transformation

The transcription antiterminator RfaH has been shown to undergo major structural rearrangements to perform multiple functions. Structural determination of the C-terminal domain (CTD) of RfaH showed that it can exist as either an α-helix bundle when interfacing with the N-terminal domain (NTD) or as a β-barrel conformation when it is not interfacing with the NTD. In this paper, we investigate the full RfaH with both CTD and NTD using a variety of all-atom molecular dynamics (MD) simulation techniques, including targeted molecular dynamics, steered molecular dynamics, and adaptive biasing force, and calculate potentials of mean force. We also use network analysis to determine communities of amino acids that are important in transferring information about structural changes. We find that the CTD–NTD interdomain interactions constitute the main barrier in the CTD α-helix to β-barrel structural conversion. Once the interfacial interactions are broken, the structural conversion of the CTD is relatively easy. We determined which amino acids play especially important roles in controlling the interdomain motions and also describe subtle structural changes that may be important in the functioning of RfaH

The Role of the Interdomain Interactions on RfaH Dynamics and Conformational Transformation

The transcription antiterminator RfaH has been shown to undergo major structural rearrangements to perform multiple functions. Structural determination of the C-terminal domain (CTD) of RfaH showed that it can exist as either an α-helix bundle when interfacing with the N-terminal domain (NTD) or as a β-barrel conformation when it is not interfacing with the NTD. In this paper, we investigate the full RfaH with both CTD and NTD using a variety of all-atom molecular dynamics (MD) simulation techniques, including targeted molecular dynamics, steered molecular dynamics, and adaptive biasing force, and calculate potentials of mean force. We also use network analysis to determine communities of amino acids that are important in transferring information about structural changes. We find that the CTD–NTD interdomain interactions constitute the main barrier in the CTD α-helix to β-barrel structural conversion. Once the interfacial interactions are broken, the structural conversion of the CTD is relatively easy. We determined which amino acids play especially important roles in controlling the interdomain motions and also describe subtle structural changes that may be important in the functioning of RfaH

In Silico Investigations of Calcium Phosphate Mineralization in Extracellular Vesicles

Calcification in bone, cartilage, and cardiovascular tissues involves the release of specialized extracellular vesicles (EVs) that promote mineral nucleation. The small size of the EVs, however, makes molecular level studies difficult, and consequently uncertainty exists on the role and function of these structures in directing mineralization. The lack of mechanistic understanding associated with the initiators of ectopic mineral deposition has severely hindered the development of potential therapeutic options. Here, we used multiscale molecular dynamics simulations to investigate the calcification within the EVs. Results show that Ca²⁺–HPO₄^2– and phosphatidylserine complexes facilitate the early nucleation. Use of coarse-grained simulations allows investigations of Ca²⁺–PO₄^3– nucleation and crystallization in the EVs. Systematic variation in the ion-to-water ratio shows that the crystallization and growth strongly depend on the enrichment of the ions and dehydration inside the EVs. Our investigations provide insights into the role of EVs on calcium phosphate mineral nucleation and growth in both physiological and pathological mineralization

Molecular Dynamics Investigations of the α‑Helix to β‑Barrel Conformational Transformation in the RfaH Transcription Factor

The C-terminal domain (CTD) of the transcription antiterminator RfaH folds to an α-helix bundle when it interacts with its N-terminal domain (NTD) but it undergoes an all-α to all-β conformational transformation when it does not interact with the NTD. The RfaH-CTD in the all-α topology is involved in regulating transcription whereas in the all-β topology it is involved in stimulating translation by recruiting a ribosome to an mRNA. Because the conformational transformation in RfaH-CTD gives it a different function, it is labeled as a transformer protein, a class that may eventually include many other functional proteins. The structure and function of RfaH is of interest for its own sake, as well as for the value it may serve as a model system for investigating structural transformations in general. We used replica exchange molecular dynamics simulations with implicit solvent to investigate the α-helix to β-structure transformation of RfaH-CTD, followed by structural relaxation with detailed all atom simulations for the best replica. The importance of interfacial interactions between the two domains of RfaH is highlighted by the compromised structural integrity of the helical form of the CTD in the absence NTD. Calculations of free-energy landscape and transfer entropy elucidate the details of the RfaH-CTD transformation process

Exploring the Diffusion of Molecular Oxygen in the Red Fluorescent Protein mCherry Using Explicit Oxygen Molecular Dynamics Simulations

The development of fluorescent proteins (FPs) has revolutionized cell biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for tagging and tracking cellular processes in vivo. Determining oxygen diffusion pathways in FPs can be important for improving photostability and for understanding maturation of the chromophore. We use molecular dynamics (MD) calculations to investigate the diffusion of molecular oxygen in one of the most useful monomeric RFPs, mCherry. We describe a pathway that allows oxygen molecules to enter from the solvent and travel through the protein barrel to the chromophore. We calculate the free-energy of an oxygen molecule at points along the path. The pathway contains several oxygen hosting pockets, which are identified by the amino acid residues that form the pocket. We also investigate an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins

Hydrogen Bond Flexibility Correlates with Stokes Shift in mPlum Variants

Fluorescent proteins have revolutionized molecular biology research and provide a means of tracking subcellular processes with extraordinary spatial and temporal precision. Species with emission beyond 650 nm offer the potential for deeper tissue penetration and lengthened imaging times; however, the origin of their extended Stokes shift is not fully understood. We employed spectrally resolved transient grating spectroscopy and molecular dynamics simulations to investigate the relationship between the flexibility of the chromophore environment and Stokes shift in mPlum. We examined excited state solvation dynamics in a panel of strategic point mutants of residues E16 and I65 proposed to participate in a hydrogen-bonding interaction thought responsible for its red-shifted emission. We observed two characteristic relaxation constants of a few picoseconds and tens of picoseconds that were assigned to survival times of direct and water-mediated hydrogen bonds at the 16-65 position. Moreover, variants of the largest Stokes shift (mPlum, I65V) exhibited significant decay on both time scales, indicating the bathochromic shift correlates with a facile switching between a direct and water-mediated hydrogen bond. This dynamic model underscores the role of environmental flexibility in the mechanism of excited state solvation and provides a template for engineering next-generation red fluorescent proteins