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How the crowded environment inside cells affects folding, stability and structures of proteins is a vital question, since most proteins are made and function inside cells. Here we describe how crowded conditions can be created in vitro and in silico and how we have used this to probe effects on protein properties. We have found that folded forms of proteins become more compact in the presence of macromolecular crowding agents; if the protein is aspherical, the shape also changes (extent dictated by native-state stability and chemical conditions). It was also discovered that the shape of the macromolecular crowding agent modulates the folding mechanism of a protein; in addition, the extent of asphericity of the protein itself is an important factor in defining its folding speed.

Proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been well established that the so-called “macromolecular crowding” effect enhances the folding stability of proteins by destabilizing their unfolded states for selected proteins. On the other hand, chemical and thermal denaturation is often used in experiments as a tool to destabilize a protein by populating the unfolded states when probing its folding landscape and thermodynamic properties. However, little is known about the complicated effects of these synergistic perturbations acting on the kinetic properties of proteins, particularly when large structural fluctuations, such as protein folding, have been involved. In this study, we have first investigated the folding mechanism of Trp-cage dependent on urea concentration by coarse-grained molecular simulations where the impact of urea is implemented into an energy function of the side chain and/or backbone interactions derived from the all-atomistic molecular dynamics simulations with urea through a Boltzmann inversion method. In urea solution, the folding rates of a model mini protein Trp-cage decrease and the folded state slightly swells due to a lack of contact formation between side chains at the terminal regions. In addition, the equilibrium m-values of Trp-cage from the computer simulations are in agreement with experimental measurements. We have further investigated the combined effects of urea denaturation and macromolecular crowding on Trp-cage's folding mechanism where crowding agents are modeled as hard-spheres. The enhancement of folding rates of Trp-cage is most pronounced by macromolecular crowding effect when the extended conformations of Trp-cast dominate at high urea concentration. Our study makes quantitatively testable predictions on protein folding dynamics in a complex environment involving both chemical denaturation and macromolecular crowding effects.

Chemical and thermal denaturation methods have been widely used to investigate folding processes of proteins in vitro. However, a molecular understanding of the relationship between these two perturbation methods is lacking. Here, we combined computational and experimental approaches to investigate denaturing effects on three structurally different proteins. We derived a linear relationship between thermal denaturation at temperature T(b) and chemical denaturation at another temperature T(u) using the stability change of a protein (ΔG). For this, we related the dependence of ΔG on temperature, in the Gibbs-Helmholtz equation, to that of ΔG on urea concentration in the linear extrapolation method, assuming that there is a temperature pair from the urea (T(u)) and the aqueous (T(b)) ensembles that produces the same protein structures. We tested this relationship on apoazurin, cytochrome c, and apoflavodoxin using coarse-grained molecular simulations. We found a linear correlation between the temperature for a particular structural ensemble in the absence of urea, T(b), and the temperature of the same structural ensemble at a specific urea concentration, T(u). The in silico results agreed with in vitro far-UV circular dichroism data on apoazurin and cytochrome c. We conclude that chemical and thermal unfolding processes correlate in terms of thermodynamics and structural ensembles at most conditions; however, deviations were found at high concentrations of denaturant.

Topological frustrated proteins can give rise to complex folding pathways and transition state profiles. To better understand such systems, we combine experimental and computational methods to study Desulfovibrio desulfuricans apoflavodoxin by producing several point mutation variants. By equilibrium unfolding experiments, we first revealed how different secondary-structure elements contribute to overall protein resistance towards heat and urea. Next using stopped-flow mixing coupled to far-UV circular dichroism (CD), we probed how individual residues affect the amount of structure formed in the experimentally-detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of the growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e. at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in the folding nucleigrowth correlates with the in vitro burst phase CD amplitude. In addition, Φ-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding while proper alignment on both sides of β-strand 3 is necessary for productive folding.

How the crowded environment inside cells affects the structures of proteins with aspherical shapes is a vital question because many proteins and protein–protein complexes in vivo adopt anisotropic shapes. Here we address this question by combining computational and experimental studies of a football-shaped protein (i.e., Borrelia burgdorferi VlsE) in crowded, cell-like conditions. The results show that macromolecular crowding affects protein-folding dynamics as well as overall protein shape. In crowded milieus, distinct conformational changes in VlsE are accompanied by secondary structure alterations that lead to exposure of a hidden antigenic region. Our work demonstrates the malleability of “native” proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo .

Chemical and thermal denaturation has been widely used in order to investigate the folding processes of proteins in vitro. However, a comprehensive understanding of relationship between chemical and thermal perturbations of proteins at molecular level has been challenging. Here, we have used combined computational and experimental approaches to investigate the denaturing effect on structurally different, unrelated proteins. We have identified a linear relationship between thermal denaturation and chemical denaturation through the stability of protein folding (ΔG) by relating the dependence of ΔG on temperature in the Gibbs-Helmholtz equation and that of ΔG on urea concentration in the linear extrapolation method. We have tested this relationship on apoazurin, cytochrome c and apoflavodoxin by examining their structural similarities of the ensembles perturbed by chemical versus thermal means using coarse-grained molecular simulations at a broad range of urea concentration and temperature. We have found that the temperature of a system in the absence of urea at Tb is linearly proportional to the temperature of the same system under low urea concentration at Tu at a slope of one. The slope of this linear correspondence deviates from one at high urea concentration. In addition, the folding route of apoflavodoxin that involves topological frustration is influenced by the presence of urea. This in silico finding has agreed with in vitro far UV circular dichorism data for apoazurin and cytochrome c. Thus, chemical and thermal unfolding processes correlate in terms of thermodynamics and structural ensembles at most conditions, but deviations occur at high concentrations of denaturant.

Proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been well established that the so-called "macromolecular crowding" effect enhances the folding stability of proteins by destabilizing their unfolded states for selected proteins. On the other hand, chemical and thermal denaturation is often used in experiments as a tool to destabilize a protein by populating the unfolded states when probing its folding landscape and thermodynamic properties. However, little is known about the complicated effects of these synergistic perturbations acting on the kinetic properties of proteins, particularly when large structural fluctuations, such as protein folding, have been involved. In this study, we have first investigated the folding mechanism of Trp-cage dependent on urea concentration by coarse-grained molecular simulations where the impact of urea is implemented into an energy function of the side chain and/or backbone interactions derived from the all-atomistic molecular dynamics simulations with urea through a Boltzmann inversion method. In urea solution, the folding rates of a model miniprotein Trp-cage decrease and the folded state slightly swells due to a lack of contact formation between side chains at the terminal regions. In addition, the equilibrium m-values of Trp-cage from the computer simulations are in agreement with experimental measurements. We have further investigated the combined effects of urea denaturation and macromolecular crowding on Trp-cage's folding mechanism where crowding agents are modeled as hard-spheres. The enhancement of folding rates of Trp-cage is most pronounced by macromolecular crowding effect when the extended conformations of Trp-cast dominate at high urea concentration. Our study makes quantitatively testable predictions on protein folding dynamics in a complex environment involving both chemical denaturation and macromolecular crowding effects.

We use computer simulation to investigate the effects of synthetic crowders on cytochrome c, a small single-domain protein with a cofactor heme. The folding energy landscape of cytochrome was computed in the presence of crowders with various sizes and shapes by using a coarse-grained protein model and the structure-based (Go-like) interactions. Our results demonstrated that given the same volume fraction of crowders, the stability of a folded protein inversely increases with the radius of crowders. In addition, a crowder with an anisotropic geometry imposes a greater stabilizing effect on the folded protein than isotropic crowders. This is in agreement with the predictions by the scaled particle theory. In addition, the distribution of contact formation between heme and cytochrome c protein was found to be varied by different types of crowders, demonstrating that the geometry of crowders may be one of the key factors for tuning heme-protein contact formation under cell-like conditions. Prospects of mixed crowders will be presented.

In the quest to develop multiscale molecular simulation methods for complex protein dynamics that fuse high-resolution and low-resolution protein representations, it is important to investigate the required information of reconstruction of all-atomistic proteins from low resolution ones with manageable uncertainly. In this paper, we introduce a robust, accurate, and fast reconstruction method (SCAAL) that produces reliable all-atomistic protein structure by taking few beads from a coarse-grained model with at least one side chain bead and one Cα bead in the backbone (Side chain-Cα Model, SCM) into accounts. Our algorithm (SCAAL) is compared with SCWRL3.0 and it excels in robustness and is more accurate in the reconstruction of large amino acids. In addition, we further test SCAAL in the reconstruction of a complete protein folding trajectory from SCM coarse-grained models. We show that the efficiency, accuracy, and robustness of SCAAL as leverage for multi-scale simulations are excellent in terms of low root mean square deviations that lie within 1A resolution.

We developed a multiscale approach (MultiSCAAL) that integrates the potential of mean force (PMF) obtained from all-atomistic molecular dynamics simulations with a knowledge-based energy function for coarse-grained molecular simulation in better exploring the energy landscape of a small protein under chemical interference such as chemical denaturation. The two key features of this scheme are the Boltzmann inversion and a protein atomistic reconstruction method we previously developed (SCAAL). Using MultiSCAAL, we were able to enhance the sampling efficiency of proteins solvated by explicit water molecules. Our method has been tested on the folding energy landscape of a small protein Trp-cage with explicit solvent under 8M urea using both the all-atomistic replica exchange molecular dynamics (AA-REMD) and MultiSCAAL. We compared computational analyses on ensemble conformations of Trp-cage with its available experimental NOE distances. The analysis demonstrated that conformations explored by MultiSCAAL better agree with the ones probed in the experiments because it can effectively capture the changes in side chain orientations that can flip out of the hydrophobic pocket in the presence of urea and water molecules. In this regard, MultiSCAAL is a promising and effective sampling scheme for investigating chemical interference which presents a great challenge when modeling protein interactions in vivo.