Internal Water Molecules Research Articles

Coupling of Retinal, Protein, and Water Dynamics in Squid Rhodopsin

The light-induced isomerization of the retinal from 11- cis to all- trans triggers changes in the conformation of visual rhodopsins that lead to the formation of the activated state, which is ready to interact with the G protein. To begin to understand how changes in the structure and dynamics of the retinal are transmitted to the protein, we performed molecular dynamics simulations of squid rhodopsin with 11- cis and all- trans retinal, and with two different force fields for describing the retinal molecule. The results indicate that structural rearrangements in the binding pocket, albeit small, propagate toward the cytoplasmic side of the protein, and affect the dynamics of internal water molecules. The sensitivity of the active-site interactions on the retinal force-field parameters highlights the coupling between the retinal molecule and its immediate protein environment.

Giant Crown‐Shaped Polytungstate Formed by Self‐Assembly of CeIII‐Stabilized Dilacunary Keggin Fragments

The largest tungstogermanate and third-largest polytungstate (see picture) was obtained by K+-directed self-assembly of three unprecedented types of [GeW10O38]12− fragments stabilized by coordination of Ce ions on the vacancies. In its Ce6O42 central ring, which displays antiferromagnetic interactions, a K+ cation is captured by internal water molecules. Gray octahedra WO6, green tetrahedra GeO4; Ce orange, K purple, O red.

Quantum Mechanical and Quantum Mechanical/Molecular Mechanical Studies of the Iron−Dioxygen Intermediates and Proton Transfer in Superoxide Reductase

Classical and quantum-chemical computations are employed to probe the reaction intermediates and proton-transfer processes in superoxide reductase (SOR) from Desulfoarculus baarsii. Ab initio studies of the SOR active site, as well as classical and QM/MM MD simulations on the overall enzymatic reaction, are performed. We explore the use of a Hubbard U correction to standard density functional theory (DFT) in order to obtain a better description of the strongly correlated d electrons in the transition-metal center. The results obtained from the standard and Hubbard-U-corrected DFT approaches are compared with those obtained using different hybrid-DFT functionals. We show that the Hubbard U correction gives a significant improvement in the description of the structural, energetic, and electronic properties of SOR. We establish that adopting the Hubbard U correction in the QM/MM approach leads to increased accuracy with essentially no additional computational cost. Our results suggest that Lys(48) is one of the likely sources of the first proton donation to the superoxide, either directly or through an interstitial water molecule. Our QM/MM calculations highlight the important role of the interactions and hydrogen-bond network created by the imidazole rings of the His ligands and the internal water molecules. Whereas the hydrogen-bonding pattern due to internal waters can facilitate the protonation event, the interactions with the His ligands and the hydrogen bonds with water can stabilize the dioxygen ligand in a side-on conformation, which, in turn, prevents the immediate proton transfer from Lys(48), as indicated by recent experimental studies.

COMPUTATIONAL DESIGN OF NORBORNANE-BASED HIV-1 PROTEASE INHIBITORS

A series of norbornane-based HIV-1 protease (PR) inhibitors are designed theoretically to displace the tetrahedrally coordinated internal water molecule that bridges inhibitor to flaps via hydrogen bonds. These designed inhibitors use the norbornenone oxygen atom to mimic this structural water molecule and contain diols to interact with the carboxylate oxygens of catalytic aspartates. The binding free energies were estimated by modified linear interaction energy approach [Zoete H, Michielin O, Karplus M, J Comput Aided Mol Des17:861, 2003], in which the binding free energy is written as a linear combination of the electrostatic interaction energy between PR and the ligand, E elec , the van der Waals interaction energy between PR and the ligand, E vdW , and the difference of the solvation free energies of the complex, the receptor, and the isolated ligand, ΔG solv . The equation obtained in previous work [Da W. Zhang, Philip Lin Huang, Sylvia Lee-Huang, John Z. H. Zhang, J Theor Comput Chem7:485, 2008] is applied directly to calculate the binding free energy of designed norbornane-based HIV-1 PR inhibitors.

Mechanism of 1H–14N cross-relaxation in immobilized proteins

A resonant enhancement of the water-1H relaxation rate at three distinct frequencies in the range 0.5–3MHz has been observed in a variety of aqueous biological systems. These so-called quadrupole (Q) peaks have been linked to a dipolar flip–flop polarization transfer from 1H nuclei to rapidly relaxing amide 14N nuclei in rotationally immobilized proteins. While the Q-peak frequencies conform to the known amide 14N quadrupole coupling parameters, a molecular model that accounts for the intensity and shape of the Q peaks has not been available. Here, we present such a model and test it against an extensive set of Q-peak data from two fully hydrated crosslinked proteins under conditions of variable temperature, pH and H/D isotope composition. We propose that polarization transfer from bulk water to amide 14N occurs in three steps: from bulk water to a so-called intermediary proton via material diffusion/exchange, from intermediary to amide proton by cross-relaxation driven by exchange-mediated orientational randomization of their mutual dipole coupling, and from amide proton to 14N by resonant dipolar relaxation ‘of the second kind’, driven by 14N spin fluctuations, which, in turn, are induced by restricted rigid-body motions of the protein. An essentially equivalent description of the last step can be formulated in terms of coherent 1H→14N polarization transfer followed by fast 14N relaxation. Using independent structural and kinetic information, we show that the Q peaks from these two proteins involve ∼7 intermediary protons in internal water molecules and side-chain hydroxyl groups with residence times of order 10−5s. The model not only accounts quantitatively for the extensive data set, but also explains why Q peaks are hardly observed from gelatin gels.

Drawing the Retinal Out of Its Comfort Zone: An ONIOM(QM/MM) Study of Mutant Squid Rhodopsin

Engineering squid rhodopsin with modified retinal analogues is essential for understanding the conserved steric and electrostatic interaction networks that govern the architecture of the Schiff base binding site. Depriving the retinal of its steric and electrostatic contacts affects the positioning of the Schiff-base relative to the key residues Asn87, Tyr111, and Glu180. Displacement of the W1 and W2 positions and the impact on the structural rearrangements near the Schiff base binding region reiterates the need for the presence of internal water molecules and the accessibility of binding sites to them. Also, the dominant role of the Glu180 counterion in inducing the S(1)/S(2) state reversal for SBR is shown for the first time in squid rhodopsin.

Effects of Sensory Rhodopsin II Complexation with its Cognate Transducer HtrII on the Local Environment of Internal Water Molecules

Recent studies indicate that internal water molecules play a critical role in membrane protein function. Here we report evidence that the local environment of one or more internal water molecules in sensory rhodopsin II (SRII) is altered by interaction with its cognate transducer HtrII. The SRII-HtrII complex mediates blue-light repellent phototaxis in halophilic archaea, using a signaling pathway similar to that in bacterial chemotaxis. We studied the photocycle of SRII and a SRII-HtrII fusion complex from Natronobacterium pharaonis using low-temperature static and room temperature time-resolved Fourier transform infrared (FTIR) difference spectroscopy. When cooled to 80 K, and illuminated the protein is trapped in its K state. A shift of 2 cm−1 between SRII (3626 cm−1) and SRII-HtrII (3628 cm−1) is found for a negative OH stretching band assigned to an internal water molecule, most likely located near the active Schiff base. In contrast, the OH stretching band for this water in the K state appears at the same frequency (3619 cm−1) for both the free and complexed receptor. Similar shifts are observed upon hydration with H2O18 shifted to a lower frequency confirming these bands arise from the OH stretching mode of water. Data are also presented on the effects of lipid environment on structural changes of internal water molecules and the receptor-transducer interactions. This work was supported by National Institutes of Health GrantsR01GM069969 (to K.J.R.) and R37GM27750 (to J.L.S.), U.S. Department of Energy Grant DE-FG02-07ER15867, and the Robert A. Welch Foundation (to J.L.S.).

The Effect of Non-Coordinated Water in the Heme Pocket on the Ligand Binding Dynamics of Heme Proteins

Water molecules in internal protein cavities play fundamental roles in satisfying the H-bonding potentials of main chain atoms in turns, coils, and loops, determining the stability and rigidity of proteins, shifting the pKa values of buried ionizable residues, and modulating dynamical processes such as folding, catalysis, and proton transfers. Detecting these internal water molecules is sometimes obscured in x-ray crystallography due to positional disorder. We have developed a spectrokinetic assay that accurately detects the presence of a non-coordinated water molecule in the distal heme pocket of myoglobin and in a series of distal pocket mutants, including many where this water molecule is positionally disordered. We also have shown that this water plays a major role in determining the observed bimolecular recombination rate constant. We show that 1) this water molecule modulates the ligand binding dynamics of a series of H64, L29 and V68 mutants; 2) it plays the major role in the observed pH dependence of the CO recombination kinetics between pH 4 and 7 with the protonation of the distal histidine acting as a switch to change water occupancy; and 3) it may also modulate ligand binding dynamics in isolated hemoglobin chains, with the occupancy being larger in the alpha chains. Accurately measuring water occupancy in heme proteins answers crucial questions about water in apolar or slightly polar protein cavities and clarifies the role internal water molecules play in modulating protein function.

The Effect of Distal Heme Pocket Mutations on the Water Accessible Areas in Myoglobin

Internal water molecules are important to protein structure and function. A non-coordinated water molecule in the distal pocket of a myoglobin has been shown to be the dominate factor in controlling the binding of CO to the heme active site. We previously developed a method to experimentally measure the entry of internal water into the distal pockets of Mb mutants after photodissociation of CO. In order to better understand what factors control the occupancy of this disordered water in the protein we compared the occupancy with the size of the mutated residue and hydrophobicity. We see little correlation between residue size and water occupancy and a good correlation between water occupancy and hydrophobicity. In order to better understand what factors contribute to internal water occupancy, we further examined how cavity volume and the dynamic behavior of the distal histidine influence water occupancy. Using a computational approach, we calculated the internal volumes of myoglobin cavities for various mutants. We further characterized these cavities by investigating the dynamic behavior of the H64 residue using molecular dynamics. The data show high flexibility of the H64 in the wild type protein suggesting a mechanism by which water is allowed access to the distal cavity. However, in the distal pocket mutants, the H64 can adopt a more stable conformation thereby reducing water access to the cavity. These findings suggest that the flexibility of the distal histidine plays a key role in influencing water access to the distal cavity and the binding affinity for gaseous ligands. In addition, the long range molecular dynamics was used to assess stability of the cavity bound water for the various mutants. The obtained data showed correlation between hydrophobicity and the water residence time in the cavity.

High water mobility on the ice-binding surface of a hyperactive antifreeze protein

Antifreeze proteins (AFPs) prevent uncontrolled ice formation in organisms exposed to subzero temperatures by binding irreversibly to specific planes of nascent ice crystals. To understand the thermodynamic driving forces and kinetic mechanism of AFP activity, it is necessary to characterize the hydration behavior of these proteins in solution. With this aim, we have studied the hyperactive insect AFP from Tenebrio molitor (TmAFP) with the (17)O magnetic relaxation dispersion (MRD) method, which selectively monitors the rotational motion and exchange kinetics of water molecules on picosecond-microsecond time scales. The global hydration behavior of TmAFP is found to be similar to non-antifreeze proteins, with no evidence of ice-like or long-ranged modifications of the solvent. However, two sets of structural water molecules, located within the core and on the ice-binding face in the crystal structure of TmAFP, may have functional significance. We find that 2 of the 5 internal water molecules exchange with a residence time of 8 +/- 1 micros at 300 K and a large activation energy of approximately 50 kJ mol(-1), reflecting intermittent large-scale conformational fluctuations in this exceptionally dense and rigid protein. Six water molecules arrayed with ice-like spacing in the central trough on the ice-binding face exchange with bulk water on a sub-nanosecond time scale. The combination of high order and fast exchange may allow these water molecules to contribute entropically to the ice-binding affinity without limiting the absorption rate.

Water and Backbone Dynamics in a Hydrated Protein

Rotational immobilization of proteins permits characterization of the internal peptide and water molecule dynamics by magnetic relaxation dispersion spectroscopy. Using different experimental approaches, we have extended measurements of the magnetic field dependence of the proton-spin-lattice-relaxation rate by one decade from 0.01 to 300 MHz for 1H and showed that the underlying dynamics driving the protein 1H spin-lattice relaxation is preserved over 4.5 decades in frequency. This extension is critical to understanding the role of 1H 2O in the total proton-spin-relaxation process. The fact that the protein-proton-relaxation-dispersion profile is a power law in frequency with constant coefficient and exponent over nearly 5 decades indicates that the characteristics of the native protein structural fluctuations that cause proton nuclear spin-lattice relaxation are remarkably constant over this wide frequency and length-scale interval. Comparison of protein-proton-spin-lattice-relaxation rate constants in protein gels equilibrated with 2H 2O rather than 1H 2O shows that water protons make an important contribution to the total spin-lattice relaxation in the middle of this frequency range for hydrated proteins because of water molecule dynamics in the time range of tens of ns. This water contribution is with the motion of relatively rare, long-lived, and perhaps buried water molecules constrained by the confinement. The presence of water molecule reorientational dynamics in the tens of ns range that are sufficient to affect the spin-lattice relaxation driven by 1H dipole-dipole fluctuations should make the local dielectric properties in the protein frequency dependent in a regime relevant to catalytically important kinetic barriers to conformational rearrangements.

Optical Detection of Disordered Water within a Protein Cavity

Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by X-ray crystallography. Here, we show that water can be detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not readily observed in the corresponding crystal structures. A well-defined, noncoordinated water molecule hydrogen bonded to the distal histidine (His64) is seen within the distal heme pocket in the crystal structure of wild type (wt) deoxymyoglobin. Displacement of this water decreases the rate of ligand entry into wt Mb, and we have shown previously that the entry of this water is readily detected optically after laser photolysis of MbCO complexes. However, for L29F and V68L Mb no discrete positions for solvent molecules are seen in the electron density maps of the crystal structures even though His64 is still present and slow rates of ligand binding indicative of internal water are observed. In contrast, time-resolved perturbations of the visible absorption bands of L29F and V68L deoxyMb generated after laser photolysis detect the entry and significant occupancy of water within the distal pockets of these variants. Thus, the spectral perturbation of pentacoordinate heme offers a potentially robust system for measuring nonspecific hydration of the active sites of heme proteins.

Properties of the Anion-Binding Site of pharaonis Halorhodopsin Studied by Ultrafast Pump−Probe Spectroscopy and Low-Temperature FTIR Spectroscopy

Halorhodopsin (HR) is a light-driven chloride pump. Cl(-) is bound in the Schiff base region of the retinal chromophore, and unidirectional Cl(-) transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. It is known that HR from Natronobacterium pharaonis (pHR) also pumps NO(3)(-) with similar efficiency, suggesting that NO(3)(-) binds to the Cl(-)-binding site. In the present study, we investigated the properties of the anion-binding site by means of ultrafast pump-probe spectroscopy and low-temperature FTIR spectroscopy. The obtained data were surprisingly similar between pHR-NO(3)(-) and pHR-Cl(-), even though the shapes and sizes of the two anions are quite different. Femtosecond pump-probe spectroscopy showed very similar excited-state dynamics between pHR-NO(3)(-) and pHR-Cl(-). Low-temperature FTIR spectroscopy of unlabeled and [zeta-(15)N]Lys-labeled pHR revealed almost identical hydrogen-bonding strengths of the protonated retinal Schiff base between pHR-NO(3)(-) and pHR-Cl(-), which is similarly strengthened after retinal isomerization. There were spectral variations for water stretching vibrations between pHR-NO(3)(-) and pHR-Cl(-), suggesting that the water molecules hydrate each anion. Nevertheless, the overall spectral features were similar for the two species. These observations strongly suggest that the anion-binding site has a flexible structure and that the interaction between retinal and the anions is weak, despite the presence of an electrostatic interaction. Such a flexible hydrogen-bonding network in the Schiff base region in HR appears to be in remarkable contrast to that in light-driven proton-pumping proteins.

Observation of “Ionic Lock” Formation in Molecular Dynamics Simulations of Wild-Type β1 and β2 Adrenergic Receptors

G protein coupled receptors (GPCRs) are a large family of integral membrane proteins involved in signal transduction pathways, making them appealing drug targets for a wide spectrum of diseases. The recently crystallized structures of two engineered adrenergic receptors have opened new avenues for the understanding of the molecular mechanisms of action of GPCRs. Taking the two crystal structures as a starting point, we carried out submicrosecond molecular dynamics simulations of wild-type beta(1) and beta(2) adrenergic receptors in a lipid bilayer under physiological conditions. These simulations give access to structural and dynamic properties of the receptors in pseudo in vivo conditions. For both systems the overall fold properties of the transmembrane region as well as the binding pocket remain close to the crystal structure of the engineered systems, thus suggesting that the ligand binding mode is not affected by the introduced modifications. Both simulations indicate the presence of one or two internal water molecules absent in both crystal structures and essential for the stabilization of the binding pocket at the interface between transmembrane helices III, IV, and V. The different interactions arising from the substitution of Tyr308 in beta(2)AR into Phe325 in beta(1)AR induce different conformations of the homologous Asn(6.55) inside the binding pockets of the two receptors, suggesting a possible origin of receptor specificity in agonist binding. The equilibrated structures of both receptors recover all of the previously suggested features of inactive GPCRs including formation of a salt bridge between the cytoplasmatic moieties of helices III and VI ("ionic lock") that is absent in the crystal structures.

Spectroscopic and Kinetic Evidence on How Bacteriorhodopsin Accomplishes Vectorial Proton Transport under Functional Conditions

Vectorial transport by pumps requires a switch in the accessibility (or affinity) of the ion binding site from the extracelullar to the cytoplamic side, or vice versa. In the proton-pump bacteriorhodopsin (bR) the nature of this switch mechanism is still controversial, although it is expected to occur during the transition between two M substates. Here, we characterized this transition by time-resolved Fourier transform infrared (FT-IR) spectroscopy under functional conditions, using a novel approach for the analysis of kinetic data: the regularized inversion of an eigenvalue problem. The use of IR spectroscopy allowed the simultaneous evaluation of the involvement of the protein backbone, retinal, amino acid side chains, and internal water molecules in the switch mechanism. We provide solid evidence that the switch is not associated with protein backbone conformational changes. On the other hand, changes in the retinal conformation (or in the orientation of the Schiff Base (SB) during the switch) are reasonably although not completely discarded. We found that the proton release group (PRG), a delocalized proton characterized by a broad continuum band in the infrared, deprotonates in the transition between two M substates. Vectorial proton transport is most likely guaranteed by the coupled proton affinity changes resulting from the PRG deprotonation, favoring an affinity-based over an accessibility-based switch mechanism.

Dynamics of the Internal Water Molecules in Squid Rhodopsin

Understanding the mechanism of G-protein coupled receptors action is of major interest for drug design. The visual rhodopsin is the prototype structure for the family A of G-protein coupled receptors. Upon photoisomerization of the covalently bound retinal chromophore, visual rhodopsins undergo a large-scale conformational change that prepares the receptor for a productive interaction with the G-protein. The mechanism by which the local perturbation of the retinal cis-trans isomerization is transmitted throughout the protein is not well understood. The crystal structure of the visual rhodopsin from squid solved recently suggests that a chain of water molecules extending from the retinal toward the cytoplasmic side of the protein may play a role in the signal transduction from the all- trans retinal geometry to the activated receptor. As a first step toward understanding the role of water in rhodopsin function, we performed a molecular dynamics simulation of squid rhodopsin embedded in a hydrated bilayer of polyunsaturated lipid molecules. The simulation indicates that the water molecules present in the crystal structure participate in favorable interactions with side chains in the interhelical region and form a persistent hydrogen-bond network in connecting Y315 to W274 via D80.

Molecular Dynamics Study of the Internal Water Molecules in Vasopressin and Oxytocin Receptors

The role of the internal water molecules in vasopressin and oxytocin receptors has been investigated via molecular dynamics simulations in hydrated membrane model. Several water molecules have been identified within the binding pockets of receptors, where they interact with the conserved residues. In all unliganded receptors, the water molecules bound to the highly conserved D2.50 cluster have been observed. It has been proposed which water molecules may significantly contribute to the stability of overall receptor structure. In receptor-ligand complexes the water molecules are involved in the receptor-ligand interactions by forming water-mediated hydrogen bonds at their contact surface.

Quantitative Analysis of Water Dynamics in and near Proteins

Proteins exist in aqueous solution. Hydration can be viewed as a description of how the protein disturbs the structure and dynamics of water. Water molecules in the vicinity of proteins are generally seen as either external or internal water molecules. Internal water molecules occupy cavities, exchange on a time-scale of 0.1--10 microseconds with bulk water, are almost as conserved as amino acids, and are therefore likely to be important for function. External water molecules tend to be found in protein crevices and are typically not conserved, even between crystal structures of the same protein. We introduce a method to analyze the behavior of water molecules in molecular dynamics (MD) simulations in terms of graphs. The graph encodes a simple hopping model: Nodes in the graph correspond to hydration sites, typically defined from the density in computer simulations or observed water sites in crystal structures. Directed edges correspond to transitions (“hops”) between sites, with transition rates computed from MD simulations. We apply this analysis to the water-filled cavity of intestinal fatty acid binding protein (I-FABP) in its apo and holo (palmitate-bound) state. This demonstrates how ligand binding influences the well-defined set of hydration sites in and around the protein's cavity. The ligand displaces a number of hydration sites but does not affect others close by. The parameters extracted from the network model allow us to model the movement of water molecules with a Markov Chain Monte Carlo model. The graphical construct reproduces the average site occupancy found in the MD simulations and the fluctuations of the occupancy. This approach suggests new types of sampling and analysis that can be applied to extend the range of molecular dynamics models and the role of water in ligand binding.

The Green-absorbing Drosophila Rh6 Visual Pigment Contains a Blue-shifting Amino Acid Substitution That Is Conserved in Vertebrates

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Through sequence analysis and functional investigation of vertebrate visual pigments, numerous amino acid substitutions important for this adaptive process have been identified. Here we describe a serine/alanine (S/A) substitution in long wavelength-absorbing Drosophila visual pigments that occurs at a site corresponding to Ala-292 in bovine rhodopsin. This S/A substitution accounts for a 10-17-nm absorption shift in visual pigments of this class. Additionally, we demonstrate that substitution of a cysteine at the same site, as occurs in the blue-absorbing Rh5 pigment, accounts for a 4-nm shift. Substitutions at this site are the first spectrally significant amino acid changes to be identified for invertebrate pigments sensitive to visible light and are the first evidence of a conserved tuning mechanism in vertebrate and invertebrate pigments of this class.

Dynamics of the Internal Water Molecules in Squid Rhodopsin

G-protein coupled receptors (GPCRs) are major parmaceutical targets because of their key role in a diverse array of physiological functions. The visual rhodopsin is the prototype for the family A of GPCRs. Upon photoisomerization of the covalently-bound retinal chromophore, visual rhodopsins undergo a large-scale conformational change that prepares the receptor for a productive interaction with the G protein. The mechanism by which the local perturbation of the retinal cis-trans isomerization is transmitted throughout the protein is not well understood. The recently reported crystal structure of squid rhodopsin (M. Murakami and T. Kouyama, Nature 453, 363, 2008) displays new features that may provide additional insight into the mechanism of the signal transduction in GPCRs. It has been suggested, based on the location of water molecules in the interhelical region extending from the retinal towards the cytoplasmic side, that a water-mediated hydrogen-bond network may play a role in the activation process. As a first step towards understanding the role of water in rhodopsin function, we have performed a molecular dynamics simulation of squid rhodopsin embedded in a hydrated bilayer of polyunsaturated lipid molecules. Here we report results from the simulation that show that the water molecules present in the crystal structure participate in favorable interactions with side chains in the interhelical region, and form a persistent hydrogen-bond network in connecting Tyr315 to Trp274 via Asp80. We also present preliminary results from a simulation study of the changes in the structure and dynamics of the hydrogen-bond network that accompany the photoisomerization of the retinal chromophore.