Pressure Adaptations in Deep-Sea Moritella Dihydrofolate Reductases: Compressibility versus Stability.

Proteins from "pressure-loving" piezophiles appear to adapt by greater compressibility via larger total cavity volume. However, larger cavities in proteins have been associated with lower unfolding pressures. Here, dihydrofolate reductase (DHFR) from a moderate piezophile Moritella profunda (Mp) isolated at ~2.9 km in depth and from a hyperpiezophile Moritella yayanosii (My) isolated at ~11 km in depth were compared using molecular dynamics simulations. Although previous simulations indicate that MpDHFR is more compressible than a mesophile DHFR, here the average properties and a quasiharmonic analysis indicate that MpDHFR and MyDHFR have similar compressibilities. A cavity analysis also indicates that the three unique mutations in MyDHFR are near cavities, although the cavities are generally similar in size in both. However, while a cleft overlaps an internal cavity, thus forming a pathway from the surface to the interior in MpDHFR, the unique residue Tyr103 found in MyDHFR forms a hydrogen bond with Leu78, and the sidechain separates the cleft from the cavity. Thus, while Moritella DHFR may generally be well suited to high-pressure environments because of their greater compressibility, adaptation for greater depths may be to prevent water entry into the interior cavities.

Adaptations for Pressure and Temperature in Dihydrofolate Reductases.

Enzymes from extremophilic microbes that live in extreme conditions are generally adapted so that they function under those conditions, although adaptations for extreme temperatures and pressures can be difficult to unravel. Previous studies have shown mutation of Asp27 in Escherichia coli dihydrofolate reductase (DHFR) to Glu27 in Moritella profunda (Mp). DHFR enhances activity at higher pressures, although this may be an adaptation for cold. Interestingly, MpDHFR unfolds at ~70 MPa, while Moritella yayanosii (My) was isolated at depths corresponding to ~110 MPa, indicating that MyDHFR might be adapted for higher pressures. Here, these adaptations are examined using molecular dynamics simulations of DHFR from different microbes in the context of not only experimental studies of activity and stability of the protein but also the evolutionary history of the microbe. Results suggest Tyr103 of MyDHFR may be an adaptation for high pressure since Cys103 in helix F of MpDHFR forms an intra-helix hydrogen bond with Ile99 while Tyr103 in helix F of MyDHFR forms a hydrogen bond with Leu78 in helix E. This suggests the hydrogen bond between helices F and E in MyDHFR might prevent distortion at higher pressures.

How adding a single methylene to dihydrofolate reductase can change its conformational dynamics.

Studies of the effects of pressure on proteins from piezophilic (pressure-loving) microbes compared with homologous proteins from mesophilic microbes have been relatively rare. Interestingly, such studies of dihydrofolate reductase show that a single-site mutation from an aspartic acid to a glutamic acid can reverse the pressure-dependent monotonic decrease in activity to that in a monotonic pressure-dependent activation. This residue is near the active site but is not thought to directly participate in the catalytic mechanism. Here, the ways that addition of one carbon to the entire protein could lead to such a profound difference in pressure effects are explored using molecular dynamics simulations. The results indicate that the glutamate changes the coupling between a helix and the ß-sheet due to the extra flexibility of the side chain, which further changes correlated motions of other regions of the protein.

Understanding how water models affect the anomalous pressure dependence of their diffusion coefficients.

The self-diffusion coefficient of water shows an anomalous increase with increasing hydrostatic pressure up to a broad maximum (PmD) near 1 kbar at 298 K, which has been attributed to pressure effects on the tetrahedral hydrogen bond network of water. Moreover, the ability of a water model to reproduce anomalous properties of water is a signature that it is reproducing the network. Here, water was simulated between 1 bar and 5 kbar using three water models, two four-site (with all charges in the molecular plane) and one single-site multipole (which accounts for out-of-molecular plane charge), that have reasonable pressure-temperature properties. For these three models, the diffusion coefficients display a maximum in the pressure dependence and the radial distribution functions show good agreement with the limited experimental structural data at high pressure that are available. In addition, a variety of properties associated with the network are examined, including hydrogen bond lifetimes and occupancies, three-body angle distributions, and tetrahedral order parameters. Results suggest that the initial increasing diffusion with pressure is because hydrogen bonds are distorted and thus weakened by pressure, but above PmD, the hydrogen bonds are weakened to the point it behaves more like a normal liquid. In other words, the PmD may be a measure of the angular strength of hydrogen bonds. In addition, since the four-site models over-predict the values of PmD while the multipole model under-predicts it, out-of-plane charge may improve four-site models.

Dynamical Model for the Counteracting Effects of Trimethylamine N-Oxide on Urea in Aqueous Solutions under Pressure.

Of cosolutes found in living cells, urea denatures and trimethylamine N-oxide (TMAO) stabilizes proteins; furthermore, these effects cancel at a 2:1 ratio of urea to TMAO. Interestingly, cartilaginous fish use urea and TMAO as osmolytes at similar ratios at the ocean surface but with increasing fractions of TMAO at increasing depths. Here, molecular dynamics simulations of aqueous solutions with different urea:TMAO ratios show that the diffusion coefficients of water in the solutions vary with pressure if the urea:TMAO ratio is constant, but strikingly, they are almost pressure independent at the ratio found in these fish as a function of depth. This suggests that this ratio may be maintaining a homeostasis of water dynamics. In addition, diffusion is determined by hydrogen-bond lifetimes of the different species in the solution. Based on these observations, a dynamical model in terms of hydrogen-bond lifetimes is developed for the hydrogen bonding propensities of cosolutes and water in an aqueous solution to proteins. This model provides an explanation for both the counteracting effects of TMAO on urea denaturation and the depth-dependent urea:TMAO ratio found in cartilaginous fish.

Molecular Dynamics and Neutron Scattering Studies of Potassium Chloride in Aqueous Solution.

Neutron diffraction with isotopic substitution (NDIS) experiments were done on both natural abundance potassium and isotopically labeled 41KCl heavy water solutions to characterize the solvent structuring around the potassium ion in water. Preliminary measurements suggested that the literature value for the coherent neutron scattering length (2.69 fm) for 41K was significantly in error. This value was remeasured using a neutron powder diffractometer and found to be 2.40 fm. This revision increases significantly the contrast between the natural abundance K and 41K by about 30% (from 1.0 to 1.3 fm). The experimentally determined structure factor of the potassium ion was then compared to that calculated from molecular dynamics (MD) simulations. Previous neutron scattering measurements of potassium gave a solvation number of 5.5 (see below). In this study, the NDIS and MD results are in good agreement and allowed us to derive a coordination number of 6.1 for water molecules and 0.8 for chloride ions around each K+ ion in 4 molal aqueous KCl solution.

Adaptations for Pressure and Temperature Effects on Loop Motion in Escherichia coli and Moritella profunda Dihydrofolate Reductase.

Determining how enzymes in piezophilic microbes function at high pressure can give insights into how life adapts to living at high pressure. Here, the effects of pressure and temperature on loop motions are compared Escherichia coli (Ec) and Moritella profunda (Mp) dihydrofolate reductase (DHFR) via molecular dynamics simulations at combinations of the growth temperature and pressure of the two organisms. Analysis indicates that a flexible CD loop in MpDHFR is an adaptation for cold because it makes the adenosine binding subdomain more flexible. Also, analysis indicates that the Thr113-Glu27 hydrogen bond in MpDHFR is an adaptation for high pressure because it provides flexibility within the loop subdomain compared to the very strong Thr113-Asp27 hydrogen bond in EcDHFR, and affects the correlation of the Met20 and GH loops. In addition, the results suggest that temperature might affect external loops more strongly while pressure might affect motion between elements within the protein.

Effects of Pressure and Temperature on the Atomic Fluctuations of Dihydrofolate Reductase from a Psychropiezophile and a Mesophile.

Determining the effects of extreme conditions on proteins from "extremophilic" and mesophilic microbes is important for understanding how life adapts to living at extremes as well as how extreme conditions can be used for sterilization and food preservation. Previous molecular dynamics simulations of dihydrofolate reductase (DHFR) from a psychropiezophile (cold- and pressure-loving), Moritella profunda (Mp), and a mesophile, Escherichia coli (Ec), at various pressures and temperatures indicate that atomic fluctuations, which are important for enzyme function, increase with both temperature and pressure. Here, the factors that cause increases in atomic fluctuations in the simulations are examined. The fluctuations increase with temperature not only because of greater thermal energy and thermal expansion of the protein but also because hydrogen bonds between protein atoms are weakened. However, the increase in fluctuations with pressure cannot be due to thermal energy, which remains constant, nor the compressive effects of pressure, but instead, the hydrogen bonds are also weakened. In addition, increased temperature causes larger increases in fluctuations of the loop regions of MpDHFR than EcDHFR, and increased pressure causes both increases and decreases in fluctuations of the loops, which differ between the two.

Dynamical Effects of Trimethylamine N-Oxide on Aqueous Solutions of Urea.

Trimethylamine N-oxide (TMAO) stabilizes protein structures, whereas urea destabilizes proteins, and their opposing effects can be counteracted at a 1:2 ratio of TMAO to urea. To investigate how they affect solution dynamics, molecular dynamics simulations have been carried out for aqueous solutions of TMAO and urea at different concentrations. In the binary solutions, urea mainly slows the diffusion of waters that are hydrogen bonded to it (i.e., hydration water), whereas TMAO dramatically slows the diffusion of both hydration water and bulk water because of long-lived TMAO-water hydrogen bonds. In the ternary solutions, because TMAO decreases the diffusion rate of bulk water, the lifetimes of not only water-water but also urea-water hydrogen bonds increase. In addition, the constant forming and breaking of short lifetime hydrogen bonds between urea and water appears to impart energy into the bulk, whereas the long lifetime hydrogen bonds between TMAO and water slows down the bulk, resulting in the compensating effects on bulk water in the ternary solution. This suggests that the counteracting effects of TMAO on urea denaturation may be both to make longer lived hydrogen bonds to water and to counter the energizing effects of urea on bulk water.

Reduction potential calculations of the Fe-S clusters in Thermus thermophilus respiratory complex I.

Respiratory complex I facilitates electron transfer from NADH to quinone over ~95 Å through a chain of seven iron-sulfur (Fe-S) clusters in the respiratory chain. In this study, the reduction potentials of the Fe-S clusters in Thermus thermophilus complex I are calculated using a Density Functional Theory + Poisson-Boltzmann method. Our results indicate that the reduction potentials are influenced by a variety of factors including the clusters being deeply buried in the complex and the protonation state of buried ionizable residues. In addition, as several of the ionizable side chains have predicted pKa values near pH 7, relatively small structural fluctuations could lead to significant (0.2 V) shifts in the reduction potential of several of the Fe-S clusters, suggesting a dynamic mechanism for electron transfer. Moreover, the method used here is a useful computational tool to study other questions about complex I. © 2019 Wiley Periodicals, Inc.

Diffusion of aqueous solutions of ionic, zwitterionic, and polar solutes.

The properties of aqueous solutions of ionic, zwitterionic, and polar solutes are of interest to many fields. For instance, one of the many anomalous properties of aqueous solutions is the behavior of water diffusion in different monovalent salt solutions. In addition, solutes can affect the stabilities of macromolecules such as proteins in aqueous solution. Here, the diffusivities of aqueous solutions of sodium chloride, potassium chloride, tri-methylamine oxide (TMAO), urea, and TMAO-urea are examined in molecular dynamics simulations. The decrease in the diffusivity of water with the concentration of simple ions and urea can be described by a simple model in which the water molecules hydrogen bonded to the solutes are considered to diffuse at the same rate as the solutes, while the remainder of the water molecules are considered to be bulk and diffuse at almost the same rate as pure water. On the other hand, the decrease in the diffusivity of water with the concentration of TMAO is apparently affected by a decrease in the diffusion rate of the bulk water molecules in addition to the decrease due to the water molecules hydrogen bonded to TMAO. In other words, TMAO enhances the viscosity of water, while urea barely affects it. Overall, this separation of water molecules into those that are hydrogen bonded to solute and those that are bulk can provide a useful means of understanding the short- and long-range effects of solutes on water.

Quasiharmonic Analysis of the Energy Landscapes of Dihydrofolate Reductase from Piezophiles and Mesophiles.

A quasiharmonic analysis (QHA) method is used to compare the potential energy landscapes of dihydrofolate reductase (DHFR) from a piezophile (pressure-loving organism), Moritella profunda (Mp), and a mesophile, Escherichia coli (Ec). The QHA method considers atomic fluctuations of the protein as motions of an atom in a local effective potential created by neighboring atoms and quantitates it in terms of effective force constants, isothermal compressibilities, and thermal expansivities. The analysis indicates that the underlying potential energy surface of MpDHFR is inherently softer than that of EcDHFR. In addition, on picosecond time scales, the energy surfaces become more similar under the growth conditions of Mp and Ec. On these time scales, DHFR behaves as expected; namely, increasing temperature makes the effective energy minimum less steep because thermal fluctuations increase the available volume, whereas increasing pressure steepens it because compression reduces the available volume. Our longer simulations show that, on nanosecond time scales, increasing temperature has a similar effect as on picosecond time scales because thermal fluctuations increase the volume even more on a longer time scale. However, these simulations also indicate that, on nanosecond time scales, pressure makes the local potential less steep, contrary to picosecond time scales. Further examination of the QHA indicates the nanosecond pressure response may originate at picosecond time scales at the exterior of the protein, which suggests that protein-water interactions may be involved. The results may lead to understanding adaptations in enzymes made by piezophiles that enable them to function at higher pressures.

Enzymes from piezophiles.

The discovery of microbial communities in extreme conditions that would seem hostile to life leads to the question of how the molecules making up these microbes can maintain their structure and function. While microbes that live under extremes of temperature have been heavily studied, those that live under extremes of pressure, or "piezophiles", are now increasingly being studied because of advances in sample collection and high-pressure cells for biochemical and biophysical measurements. Here, adaptations of enzymes in piezophiles against the effects of pressure are discussed in light of recent experimental and computational studies. However, while concepts from studies of enzymes from temperature extremophiles can provide frameworks for understanding adaptations by piezophile enzymes, the effects of temperature and pressure on proteins differ in significant ways. Thus, the state of the knowledge of adaptation in piezophile enzymes is still in its infancy and many more experiments and computational studies on different enzymes from a variety of piezophiles are needed.

Building better water models using the shape of the charge distribution of a water molecule.

The unique properties of liquid water apparently arise from more than just the tetrahedral bond angle between the nuclei of a water molecule since simple three-site models of water are poor at mimicking these properties in computer simulations. Four- and five-site models add partial charges on dummy sites and are better at modeling these properties, which suggests that the shape of charge distribution is important. Since a multipole expansion of the electrostatic potential describes a charge distribution in an orthogonal basis set that is exact in the limit of infinite order, multipoles may be an even better way to model the charge distribution. In particular, molecular multipoles up to the octupole centered on the oxygen appear to describe the electrostatic potential from electronic structure calculations better than four- and five-site models, and molecular multipole models give better agreement with the temperature and pressure dependence of many liquid state properties of water while retaining the computational efficiency of three-site models. Here, the influence of the shape of the molecular charge distribution on liquid state properties is examined by correlating multipoles of non-polarizable water models with their liquid state properties in computer simulations. This will aid in the development of accurate water models for classical simulations as well as in determining the accuracy needed in quantum mechanical/molecular mechanical studies and ab initio molecular dynamics simulations of water. More fundamentally, this will lead to a greater understanding of how the charge distribution of a water molecule leads to the unique properties of liquid water. In particular, these studies indicate that p-orbital charge out of the molecular plane is important.

Quasiharmonic analysis of protein energy landscapes from pressure-temperature molecular dynamics simulations.

Positional fluctuations of an atom in a protein can be described as motion in an effective local energy minimum created by the surrounding protein atoms. The dependence of atomic fluctuations on both temperature (T) and pressure (P) has been used to probe the nature of these minima, which are generally described as harmonic in experiments such as x-ray crystallography and neutron scattering. Here, a quasiharmonic analysis method is presented in which the P-T dependence of atomic fluctuations is in terms of an intrinsic isobaric thermal expansivity αP and an intrinsic isothermal compressibility κT. The method is tested on previously reported mean-square displacements from P-T molecular dynamics simulations of lysozyme, which were interpreted to have a pressure-independent dynamical transition Tg near 200 K and a change in the pressure dependence near 480 MPa. Our quasiharmonic analysis of the same data shows that the P-T dependence can be described in terms of αP and κT where below Tg, the temperature dependence is frozen at the Tg value. In addition, the purported transition at 480 MPa is reinterpreted as a consequence of the pressure dependence of Tg and the quasiharmonic frequencies. The former also indicates that barrier heights between substates are pressure dependent in these data. Furthermore, the insights gained from this quasiharmonic analysis, which was of the energy landscape near the native state of a protein, suggest that similar analyses of other simulations may be useful in understanding such phenomena as pressure-induced protein unfolding.

Extreme biophysics: Enzymes under pressure.

A critical question about piezophilic (pressure-loving) microbes is how their constituent molecules maintain function under high pressure. Here, factors are examined that may lead to the increased activity under pressure in dihydrofolate reductase from the piezophilic Moritella profunda compared to the homologous enzyme from the mesophilic Escherichia coli. Molecular dynamics simulations are performed at various temperatures and pressures to examine how pressure affects the flexibility of the enzymes from these two microbes, since both stability and flexibility are necessary for enzyme activity. The results suggest that collective motions on the 10-ns timescale are responsible for the flexibility necessary for "corresponding states" activity at the growth conditions of the parent organism. In addition, the results suggest that while the lower stability of many enzymes from deep-sea microbes may be an adaptation for greater flexibility at low temperatures, high pressure may enhance their adaptation to low temperatures. © 2017 Wiley Periodicals, Inc.

What makes proteins work: exploring life in P-T-X.

Although considerable progress has been made in the molecular biophysics of proteins, it is still not possible to reliably design an enzyme for a given function. The current understanding of enzyme function is that both structure and flexibility are important. Much attention has been focused recently on protein folding and thus structure, spurred on by insights from the folding funnel concept. For experimental studies of protein folding, variations in temperature (T) and chemical composition (X) of the solution have been traditionally exploited, although more recent studies using variations in pressure (P) made possible through new instrumentation have led to a deeper understanding of the energy landscape of protein folding. Other work has shown that flexibility is also essential for enzymes, although it is still not clear what type is important. Another avenue has been to take advantage of 'Nature's laboratory' by exploring homologous proteins from organisms that live in extreme conditions, or 'extremophiles'. While the most studied extremophiles live at extremes of T and X, recent exploration of deep-sea environments has led to the discovery of organisms living under high P, or 'piezophiles'. An exploration of targeted enzymes from organisms with various P-T-X growth conditions coupled with advances in biophysical instrumentation and computer simulations that allow studies of these enzymes at different P-T-X conditions may lead to a better understanding of 'flexibility' and to general design criteria for active enzymes. Preface. Kamal Shukla's great contribution to science has been his vision that physical sciences could bring new insights to biological sciences, and that the marriage of methodologies, particularly theoretical/computational with experimental, was needed to tackle the complexities of biology. Furthermore, his openness to new methods and different ideas outside the current fad has helped make his vision a reality. In my remarks below, I have not tried to limit myself to projects that I know Kamal had sponsored, nor have I tried to highlight all that he has sponsored. Instead, everything I mention has been influenced directly or indirectly by his efforts. Perhaps the indirect influences are most telling, because they would not have happened without Kamal.

A single-site multipole model for liquid water.

Accurate and efficient empirical potential energy models that describe the atomistic interactions between water molecules in the liquid phase are essential for computer simulations of many problems in physics, chemistry, and biology, especially when long length or time scales are important. However, while models with non-polarizable partial charges at four or five sites in a water molecule give remarkably good values for certain properties, deficiencies have been noted in other properties and increasing the number of sites decreases computational efficiency. An alternate approach is to utilize a multipole expansion of the electrostatic potential due to the molecular charge distribution, which is exact outside the charge distribution in the limits of infinite distances or infinite orders of multipoles while partial charges are a qualitative representation of electron density as point charges. Here, a single-site multipole model of water is presented, which is as fast computationally as three-site models but is also more accurate than four- and five-site models. The dipole, quadrupole, and octupole moments are from quantum mechanical-molecular mechanical calculations so that they account for the average polarization in the liquid phase, and represent both the in-plane and out-of-plane electrostatic potentials of a water molecule in the liquid phase. This model gives accurate thermodynamic, dynamic, and dielectric properties at 298 K and 1 atm, as well as good temperature and pressure dependence of these properties.

Protein dynamics and the all-ferrous [Fe4 S4 ] cluster in the nitrogenase iron protein.

In nitrogen fixation by Azotobacter vinelandii nitrogenase, the iron protein (FeP) binds to and subsequently transfers electrons to the molybdenum-FeP, which contains the nitrogen fixation site, along with hydrolysis of two ATPs. However, the nature of the reduced state cluster is not completely clear. While reduced FeP is generally thought to contain an [Fe4 S4 ](1+) cluster, evidence also exists for an all-ferrous [Fe4 S4 ](0) cluster. Since the former indicates a single electron is transferred per two ATPs hydrolyzed while the latter indicates two electrons could be transferred per two ATPs hydrolyzed, an all-ferrous [Fe4 S4 ](0) cluster in FeP is potenially two times more efficient. However, the 1+/0 reduction potential has been measured in the protein at both 460 and 790 mV, causing the biological significance to be questioned. Here, "density functional theory plus Poisson Boltzmann" calculations show that cluster movement relative to the protein surface observed in the crystal structures could account for both measured values. In addition, elastic network mode analysis indicates that such movement occurs in low frequency vibrations of the protein, implying protein dynamics might lead to variations in reduction potential. Furthermore, the different reductants used in the conflicting measurements of the reduction potential could be differentially affecting the protein dynamics. Moreover, even if the all-ferrous cluster is not the biologically relevant cluster, mutagenesis to stabilize the conformation with the more exposed cluster may be useful for bioengineering more efficient enzymes.

Molecular Multipole Potential Energy Functions for Water.

Water is the most common liquid on this planet, with many unique properties that make it essential for life as we know it. These properties must arise from features in the charge distribution of a water molecule, so it is essential to capture these features in potential energy functions for water to reproduce its liquid state properties in computer simulations. Recently, models that utilize a multipole expansion located on a single site in the water molecule, or "molecular multipole models", have been shown to rival and even surpass site models with up to five sites in reproducing both the electrostatic potential around a molecule and a variety of liquid state properties in simulations. However, despite decades of work using multipoles, confusion still remains about how to truncate the multipole expansions efficiently and accurately. This is particularly important when using molecular multipole expansions to describe water molecules in the liquid state, where the short-range interactions must be accurate, because the higher order multipoles of a water molecule are large. Here, truncation schemes designed for a recent efficient algorithm for multipoles in molecular dynamics simulations are assessed for how well they reproduce results for a simple three-site model of water when the multipole moments and Lennard-Jones parameters of that model are used. In addition, the multipole analysis indicates that site models that do not account for out-of-plane electron density overestimate the stability of a non-hydrogen-bonded conformation, leading to serious consequences for the simulated liquid.