Identifying the minimal sets of distance restraints for FRET-assisted protein structural modeling.

Proteins naturally occur in crowded cellular environments and interact with other proteins, nucleic acids, and organelles. Since most previous experimental protein structure determination techniques require that proteins occur in idealized, non-physiological environments, the effects of realistic cellular environments on protein structure are largely unexplored. Recently, Förster resonance energy transfer (FRET) has been shown to be an effective experimental method for investigating protein structure in vivo. Inter-residue distances measured in vivo can be incorporated as restraints in molecular dynamics (MD) simulations to model protein structural dynamics in vivo. Since most FRET studies only obtain inter-residue separations for a small number of amino acid pairs, it is important to determine the minimum number of restraints in the MD simulations that are required to achieve a given root-mean-square deviation (RMSD) from the experimental structural ensemble. Further, what is the optimal method for selecting these inter-residue restraints? Here, we implement several methods for selecting the most important FRET pairs and determine the number of pairs Nr that are needed to induce conformational changes in proteins between two experimentally determined structures. We find that enforcing only a small fraction of restraints, Nr/N≲0.08, where N is the number of amino acids, can induce the conformational changes. These results establish the efficacy of FRET-assisted MD simulations for atomic scale structural modeling of proteins in vivo. Significance: Determining protein structure in vivo is essential for understanding protein function. Most protein structures have been studied in non-physiological conditions using x-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. Thus, we do not know whether the cellular environment significantly affects protein structure. We emphasize the benefits of FRET-assisted molecular dynamics simulations in characterizing protein structure in vivo at the atomic scale. We identify the minimum number of FRET pairs that can induce conformational changes in several proteins, including one that has been characterized using in-cell NMR.

SPANA: Spatial decomposition analysis for cellular-scale molecular dynamics simulations.

The rapid increase in computational power with the latest supercomputers has enabled atomistic molecular dynamics (MDs) simulations of biomolecules in biological membrane, cytoplasm, and other cellular environments. These environments often contain a million or more atoms to be simulated simultaneously. Therefore, their trajectory analyses involve heavy computations that can become a bottleneck in the computational studies. Spatial decomposition analysis (SPANA) is a set of analysis tools in the Generalized-Ensemble Simulation System (GENESIS) software package that can carry out MD trajectory analyses of large-scale biological simulations using multiple CPU cores in parallel. SPANA applies the spatial decomposition of a large biological system to distribute structural and dynamical analyses into individual CPU cores, which reduces the computational time and the memory size, significantly. SPANA opens new possibilities for detailed atomistic analyses of biomacromolecules as well as solvent water molecules, ions, and metabolites in MD simulation trajectories of very large biological systems containing more than millions of atoms in cellular environments.

Measuring Structural Changes in Cytochrome c under Crowded Conditions Using In Vitro and In Silico Approaches.

It is known from in vitro studies that macromolecular crowding in the cell effects protein structure, stability and function; but predictive studies are relatively unexplored. There are few reports where the effect of various crowder mixtures has been exploited to discern their combined effect on the structural stability of proteins. These studies are more significant because their effect can mimicked with in vivo conditions, where the environment is heterogeneous. Effects of two crowders, polyethylene glycol (PEG 400 Da), and its monomer ethylene glycol (EG) alone and in mixture on the structural stability of cytochrome c (cyt c) were determined using various spectroscopic and bioinformatics tools. The main conclusions of our study are (i) the monomer EG has a kosmotropic effect on the protein (stabilizes the protein), and has no significant effect on the tertiary structure; (ii) PEG 400 destabilizes the structure as well as the stability of the protein; and (iii) EG counteracts the destabilizing effect of PEG 400. From this investigation, it seems evident that proteins may fold or unfold in the crowded environment of the cell where various interactions assist them to maintain their structure for their functions. Bioinformatics approaches were also used to support all of the in vitro observations. Cyt c is functional protein; if the structure of the protein is modulated due to change in the environment its nature of function will also change. Our research addresses the question by modulating the environment around the protein, and the macromolecule (protein) conformation dynamics and interaction study via in vitro and in silico approaches which indirectly compares with that of the environment in-cellular milieu, which is highly crowded.

Probing molecular crowding in compressed tissues with Brillouin light scattering.

Volume regulation is key in maintaining important tissue functions, such as growth or healing. This is achieved by modulation of active contractility as well as water efflux that changes molecular crowding within individual cells. Local sensors have been developed to monitor stresses or forces in model tissues, but these approaches do not capture the contribution of liquid flows to volume regulation. Here, we use a tool based on Brillouin light scattering (BLS) that uses the interaction of a laser light with inherent picosecond timescale density fluctuations in the sample. To investigate volume variations, we induced osmotic perturbations with a polysaccharide osmolyte, Dextran (Dx), and compress cells locally within multicellular spheroids (MCSs). During osmotic compressions, we observe an increase in the BLS frequency shift that reflects local variations in the compressibility. To elucidate these data, we propose a model based on a mixing law that describes the increase of molecular crowding upon reduction of the intracellular fluids. Comparison with the data suggests a nonlinear increase of the compressibility due to the dense crowding that induces hydrodynamic interactions between the cellular polymers.

On the osmotic pressure of cells.

The chemical potential of water () provides an essential thermodynamic characterization of the environment of living organisms, and it is of equal significance as the temperature. For cells, is conventionally expressed in terms of the osmotic pressure (πosm). We have previously suggested that the main contribution to the intracellular πosm of the bacterium E. coli is from soluble negatively-charged proteins and their counter-ions. Here, we expand on this analysis by examining how evolutionary divergent cell types cope with the challenge of maintaining πosm within viable values. Complex organisms, like mammals, maintain constant internal πosm ≈ 0.285 osmol, matching that of 0.154 M NaCl. For bacteria it appears that optimal growth conditions are found for similar or slightly higher πosm (0.25-0.4 osmol), despite that they represent a much earlier stage in evolution. We argue that this value reflects a general adaptation for optimising metabolic function under crowded intracellular conditions. Environmental πosm that differ from this optimum require therefore special measures, as exemplified with gram-positive and gram-negative bacteria. To handle such situations, their membrane encapsulations allow for a compensating turgor pressure that can take both positive and negative values, where positive pressures allow increased frequency of metabolic events through increased intracellular protein concentrations. A remarkable exception to the rule of 0.25-0.4 osmol, is found for halophilic archaea with internal πosm ≈ 15 osmol. The internal organization of these archaea differs in that they utilize a repulsive electrostatic mechanism operating only in the ionic-liquid regime to avoid aggregation, and that they stand out from other organisms by having no turgor pressure.

Structural Refolding and Thermal Stability of Myoglobin in the Presence of Mixture of Crowders: Importance of Various Interactions for Protein Stabilization in Crowded Conditions.

The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how proteins fold and stay functional in such crowded conditions is essential. Several in vitro experiments have looked into the effects of macromolecular crowding on different proteins. However, there are hardly any reports regarding small molecular crowders used alone and in mixtures to observe their effects on the structure and stability of the proteins, which mimics of the cellular conditions. Here we investigate the effect of different mixtures of crowders, ethylene glycol (EG) and its polymer polyethylene glycol (PEG 400 Da) on the structural and thermal stability of myoglobin (Mb). Our results show that monomer (EG) has no significant effect on the structure of Mb, while the polymer disrupts its structure and decreases its stability. Conversely, the additive effect of crowders showed structural refolding of the protein to some extent. Moreover, the calorimetric binding studies of the protein showed very weak interactions with the mixture of crowders. Usually, we can assume that soft interactions induce structural perturbations while exclusion volume effects stabilize the protein structure; therefore, we hypothesize that under in vivo crowded conditions, both phenomena occur and maintain the stability and function of proteins.