A molecular dynamics analysis of protein structural elements

The relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, beta-sheet, and loop structural elements in free and substrate-bound hen egg-white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and beta-sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of the loop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate-bound simulation. A loop region (residues 101-107) of the active site cleft had significantly reduced motion due to interactions with the substrate. Moreover, part of a loop and a 3(10) helix (residues of 67-88) not in contact with the substrate showed a marked increase in fluctuations. That these differences in dynamics of free and substrate-bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuations of the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain phi and psi angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place either abruptly, in 1 psec or less, or with a diffusive character over 5-10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 A rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface area and the dynamics of the different structural elements.     

Evidence of Functional Protein Dynamics from X-Ray Crystallographic Ensembles

It is widely recognized that representing a protein as a single static conformation is inadequate to describe the dynamics essential to the performance of its biological function. We contrast the amino acid displacements below and above the protein dynamical transition temperature, T_D∼215K, of hen egg white lysozyme using X-ray crystallography ensembles that are analyzed by molecular dynamics simulations as a function of temperature. We show that measuring structural variations across an ensemble of X-ray derived models captures the activation of conformational states that are of functional importance just above T_D, and they remain virtually identical to structural motions measured at 300K. Our results highlight the ability to observe functional structural variations across an ensemble of X-ray crystallographic data, and that residue fluctuations measured in MD simulations at room temperature are in quantitative agreement with the experimental observable.     

Molecular dynamics studies of a DNA-binding protein: 1. A comparison of the trp repressor and trp aporepressor aqueous simulations.

The results of two 30-ps molecular dynamics simulations of the trp repressor and trp aporepressor proteins are presented in this paper. The simulations were obtained using the AMBER molecular mechanical force field and in both simulations a 6-A shell of TIP3P waters surrounded the proteins. The trp repressor protein is a DNA-binding regulatory protein and it utilizes a helix-turn-helix (D helix-turn-E helix) motif to interact with DNA. The trp aporepressor, lacking two molecules of the L-tryptophan corepressor, cannot bind specifically to DNA. Our simulations show that the N- and C-termini and the residues in and near the helix-turn-helix motifs are the most mobile regions of the proteins, in agreement with the X-ray crystallographic studies. Our simulations also find increased mobility of the residues in the turn-D helix-turn regions of the proteins. We find the average distance separating the DNA-binding motifs to be larger in the repressor as compared to the aporepressor. In addition to examining the protein residue fluctuations and deviations with respect to X-ray structures, we have also focused on backbone dihedral angles and corepressor hydrogen-bonding patterns in this paper.     

Computational studies of molecular machines: the ribosome

The past decade has produced an avalanche of experimental data on the structure and dynamics of the ribosome. Groundbreaking studies in structural biology and kinetics have placed important constraints on ribosome structural dynamics. However, a gulf remains between static structures and time dependent data. In particular, X-ray crystallography and cryo-EM studies produce static models of the ribosome in various states, but lack dynamic information. Single molecule studies produce information on the rates of transitions between these states but do not have high-resolution spatial information. Computational studies have aided in bridging this gap by providing atomic resolution simulations of structural fluctuations and transitions between configurations.     

Vicinal coupling constants and protein dynamics

The effects of motional averaging on the analysis of vicinal spin-spin coupling constants derived from proton NMR studies of proteins have been examined. Trajectories obtained from molecular dynamics simulations of bovine pancreatic trypsin inhibitor and of hen egg white lysozyme were used in conjunction with an expression for the dependence of the coupling constant on the intervening dihedral angle to calculate the time-dependent behavior of the coupling constants. Despite large fluctuations, the time-average values of the coupling constants are not far from those computed for the average structure in the cases where fluctuations occur about a single potential well. The calculated differences show a high correlation with the variation in the magnitude of the fluctuations of individual dihedral angles. For the cases where fluctuations involve multiple sites, large differences are found between the time-average values and the average structure values for the coupling constants. Comparison of the simulation results with the experimental trends suggests that side chains with more than one position are more common in proteins than is inferred from X-ray results. It is concluded that for the main chain, motional effects do not introduce significant errors where vicinal coupling constants are used in structure determinations; however, for side chains, the motional average can alter deductions about the structure. Accurately measured coupling constants are shown to provide information concerning the magnitude of dihedral angle fluctuations.     

Molecular dynamics simulations of ribonuclease T1: comparison of the free enzyme and the 2' GMP-enzyme complex

Molecular dynamics simulations were performed on free RNase T1 and the 2'GMP-RNase T1 complex in vacuum and with water in the active site along with crystallographically identified waters, allowing analysis of both active site and overall structural and dynamics changes due to the presence of 2'GMP. Differences in the active site include a closing in the presence of 2'GMP, which is accompanied by a decrease in mobility of active site residues. The functional relevance of the active site fluctuations is discussed. 2'GMP alters the motion of Tyr-45, suggesting a role for that residue in providing a hydrophobic environment for the protein-nucleic acid interactions responsible for the specificity of RNase T1. The presence of 2'GMP causes a structural change of the C-terminus of the alpha-helix, indicating the transmission of structural changes from the active site through the protein matrix. Overall fluctuations of both the free and 2'GMP enzyme forms are in good agreement with X-ray temperature factors. The motion of Trp-59 is influenced by 2'GMP, indicating differences in enzyme dynamics away from the active site, with the calculated changes following those previously seen in time-resolved fluorescence experiments.     

The nature of folded states of globular proteins.

We suggest, using dynamical simulations of a simple heteropolymer modelling the alpha-carbon sequence in a protein, that generically the folded states of globular proteins correspond to statistically well-defined metastable states. This hypothesis, called the metastability hypothesis, states that there are several free energy minima separated by barriers of various heights such that the folded conformations of a polypeptide chain in each of the minima have similar structural characteristics but have different energies from one another. The calculated structural characteristics, such as bond angle and dihedral angle distribution functions, are assumed to arise from only those configurations belonging to a given minimum. The validity of this hypothesis is illustrated by simulations of a continuum model of a heteropolymer whose low temperature state is a well-defined beta-barrel structure. The simulations were done using a molecular dynamics algorithm (referred to as the "noisy" molecular dynamics method) containing both friction and noise terms. It is shown that for this model there are several distinct metastable minima in which the structural features are similar. Several new methods of analyzing fluctuations in structures belonging to two distinct minima are introduced. The most notable one is a dynamic measure of compactness that can in principle provide the time required for maximal compactness to be achieved. The analysis shows that for a given metastable state in which the protein has a well-defined folded structure the transition to a state of higher compactness occurs very slowly, lending credence to the notion that the system encounters a late barrier in the process of folding to the most compact structure. The examination of the fluctuations in the structures near the unfolding----folding transition temperature indicates that the transition state for the unfolding to folding process occurs closer to the folded state.     

Homology modelling and molecular dynamics simulations: comparative studies of human aquaporin-1

The structures of the mammalian water transport protein Aqp1 and of its bacterial homologue GlpF enables us to test whether homology models can be used to explore relationships between structure, dynamics and function in mammalian transport proteins. Molecular dynamics simulations (totalling almost 40 ns) were performed starting from: the X-ray structure of Aqp1; a homology model of Aqp1 based on the GlpF structure; and intermediate resolution structures of Aqp1 derived from electron microscopy. Comparisons of protein RMSDs vs. time suggest that the homology models are of comparable conformational stability to the X-ray structure, whereas the intermediate resolution structures exhibit significant conformation drift. For simulations based on the X-ray structure and on homology models, the flexibility profile vs. residue number correlates well with the crystallographic B-values for each residue. In the simulations based on intermediate resolution structures, mobility of the highly conserved NPA loops is substantially higher than in the simulations based on the X-ray structure or the homology models. Pore radius profiles remained relatively constant in the X-ray and homology model simulations but showed substantial fluctuations (reflecting the higher NPA loop mobility) in the intermediate resolution simulations. The orientation of the dipoles of water molecules within the pore is of key importance in maintaining low proton permeability through Aqp1. This property seems to be quite robust to the starting model used in the simulation. These simulations suggest that homology models based on bacterial homologues may be used to derive functionally relevant information on the structural dynamics of mammalian transport proteins.     

Molecular dynamics of ferrocytochrome c. Magnitude and anisotropy of atomic displacements

Two computer simulations of the atomic motion in tuna ferrocytochrome c have been carried out. The average structures and the structural correlations of the magnitudes of the atomic position fluctuations are in substantial agreement with recent X-ray diffraction results, particularly for the protein interior. The simulations show, however, that the atomic displacements are quite anisotropic. The degree of anisotropy and the preferred directions of atomic displacement exhibit correlations with structural features of the protein.     

Temperature-dependent molecular dynamics and restrained X-ray refinement simulations of a Z-DNA hexamer

Molecular dynamics simulations of the Z-DNA hexamer 5BrdC-dG-5BrdC-dG-5BrdC-dG were performed at several temperatures between 100 K and 300 K. Above 250 K, a strong sequence-dependent flexibility in the nucleic acid is observed, with the guanine sugar and the phosphate of GpC sequences much more mobile than the cytosine sugar and phosphate of CpG sequences. At 300 K, the hexamer is in dynamic equilibrium between several Z forms, including the crystallographically determined ZI and ZII forms. The local base-pair geometry, however, is not very variable, except for the roll of the base-pairs. The hexamer molecular dynamics trajectories have been used to test the restrained parameter crystallographic refinement model for nucleic acids. X-ray diffraction intensities corresponding to observed diffraction data were computed. The average structures obtained from the simulations were then refined against the calculated intensities, using a restrained least-squares program developed for nucleic acids in order to analyse the effects of the refinement model on the derived quantities. In general, the temperature dependence of the atomic fluctuations determined directly from the refined Debye-Waller factors is in reasonably good agreement with the results obtained by calculating the atomic fluctuations directly from the Z-DNA molecular dynamics trajectories. The agreement is best for refinement of temperature factors without restraints. At the highest temperature studied (300 K), the effect of the refinement on the most mobile atoms (phosphates) is to significantly reduce the mean-square atomic fluctuations estimated from the refined Debye-Waller factors below the actual values (less than (delta r)2 greater than congruent to 0.5 A2). Analysis of the temperature-dependence of the mean-square atomic fluctuations provides information concerning the conformational potential within which the atoms move. The calculated temperature-dependence and anharmonicity of the Z-DNA helix are compared with the results observed for proteins. The average structures from the simulations were refined against the experimental X-ray intensities. It is found that low-temperature molecular dynamics simulations provide a useful tool for optimizing the refinement of X-ray structures.     

Computational investigations of structural changes resulting from point mutations in a collagen-like peptide

The results of 0.5-1.0 ns molecular dynamics simulations of the collagen-like peptides [(POG)4(POA)(POG)4]3 and [(POG)9]3 (POG: proline-hydroxyproline-glycine) are presented. All simulations were performed using the AMBER-94 molecular mechanical force field with a shell of TIP3P waters surrounding the peptides. The initial geometries for the collagen-like peptides included an x-ray crystallographic structure, a computer-generated structure, a [(POG)9]3 structure modeled from the x-ray structure, and the x-ray structure with crystallographic waters replaced with a shell of modeled TIP3P waters. We examined the molecular dynamics peptide residue rms deviation fluctuations, dihedral angles, molecular and chain end-to-end distances, helical parameters, and peptide-peptide and peptide-solvent hydrogen-bonding patterns. Our molecular dynamics simulations of [(POG)4(POA)(POG)4]3 show average structures and internal coordinates similar to the x-ray crystallographic structure. Our results demonstrate that molecular dynamics can be used to reproduce the experimental structures of collagen-like peptides. We have demonstrated the feasibility of using the AMBER-94 molecular mechanical force field, which was parameterized to model nucleic acids and globular proteins, for fibril proteins. We provide a new interpretation of peptide-solvent hydrogen bonding and a peptide-peptide hydrogen bonding pattern not previously reported in x-ray studies. Last, we report on the differences; in particular with respect to main-chain dihedral angles and hydrogen bonding, between the native and mutant collagen-like peptides.     

Receptor rigidity and ligand mobility in trypsin-ligand complexes

The trypsin-like serine proteases comprise a structurally similar family of proteins with a wide diversity of biological functions. Members of this family play roles in digestion, hemostasis, immune responses, and cancer metastasis. Bovine trypsin is an archetypical member of this family that has been extensively characterized both functionally and structurally, and that preferentially hydrolyzes Arg/Lys-Xaa peptide bonds. We have used molecular dynamics (MD) simulations to study bovine trypsin complexed with the two noncovalent small-molecule ligands, benzamidine and tranylcypromine, that have the same hydrogen-bond donating moieties as Arg and Lys side-chains, respectively. Multiple (10) simulations ranging from 1 ns to 2.2 ns, with explicit water molecules and periodic boundary conditions, were performed. The simulations reveal that the trypsin binding pocket residues are relatively rigid regardless of whether there is no ligand, a high-affinity ligand (benzamidine), or a low-affinity ligand (tranylcypromine). The thermal average of the conformations sampled by benzamidine bound to trypsin is planar and consistent with the planar internal geometry of the benzamidine crystallographic model coordinates. However, the most probable bound benzamidine conformations are +/-25 degrees out of plane, implying that the observed X-ray electron density represents an average of densities from two mirror symmetric, nonplanar conformations. Solvated benzamidine has free energy minima at +/-45 degrees , and the induction of a more planar geometry upon binding is associated with approximately 1 kcal/mol of intramolecular strain. Tranylcypromine's hydrogen-bonding pattern in the MD differs substantially from that inferred from the X-ray electron density. Early in simulations of this system, tranylcypromine adopts an alternative binding conformation, changing from the crystallographic conformation, with a direct hydrogen bond between its amino moiety and the backbone oxygen of Gly219, to one having a bridging water molecule. This result is consistently seen with the CHARMM22, Amber, or OPLS-AA force fields. The trypsin-tranylcypromine hydrogen-bonding pattern observed in the simulations also occurs as the crystallographic binding mode of the Lys15 side-chain of bovine pancreatic trypsin inhibitor bound to trypsin. In this latter cocrystal, a bridging crystallographic water does reside between the side-chain's amino group and the trypsin Gly219 backbone oxygen. Furthermore, the trypsin-tranylcypromine simulations sample two different stable noncrystallographic binding poses. These data suggest that some of the electron density ascribed to tranylcypromine in the X-ray model is rather due to a bound water molecule, and that multiple tranylcypromine binding conformations (crystallographic disorder) may be the cause of ambiguous electron density. The combined trypsin-benzamidine and trypsin- tranylcypromine results highlight the ability of simulations to augment protein-ligand complex structural data by deconvoluting the effects of thermal and structural averaging, and by finding energetically optimal ligand and bound water positions for weakly bound ligands.     

Structure and internal mobility of proteins: a molecular dynamics study of hen egg white lysozyme

The structure and internal motions of the protein hen egg white lysozyme are studied by analysis of simulation and experimental data. A molecular dynamics simulation and an energy minimization of the protein in vacuum have been made and the results compared with high-resolution structures and temperature factors of hen egg white lysozyme in two different crystal forms and of the homologous protein human lysozyme. The structures obtained from molecular dynamics and energy minimization have root-mean-square deviations for backbone atoms of 2.3 Å and 1.1–1.3 Å, respectively, relative to the crystal structures; the different crystal structures have root-mean-square deviations of 0.73–0.81 Å for the backbone atoms. In comparing the backbone dihedral angles, the difference between the dynamics and the crystal structure on which it is based is the same as that between any two crystal structures. The internal fluctuations of atomic positions calculated from the molecular dynamics trajectory agree well with the temperature factors from the three structures. Simulation and crystal results both show that there are large motions for residues involved in exposed turns of the backbone chain, relatively smaller motions for residues involved in the middle of helices or β-sheet structures, and relatively small motions of residues near disulfide bridges. Also, both the simulation and crystal data show that side-chain atoms have larger fluctuations than main-chain atoms. Moreover, the regions that have large deviations among the x-ray crystal structures, which indicates flexibility, are found to have large fluctuations in the simulation.     

Free energy simulations for protein ligand binding and stability

Abstract

We summarize several computational techniques to determine relative free energies for condensed-phase systems. The focus is on practical considerations which are capable of making direct contact with experiments. Particular applications include the thermodynamic stability of apo- and holo-myoglobin, insulin dimerization free energy, ligand binding in lysozyme, and ligand diffusion in globular proteins. In addition to provide differential free energies between neighboring states, converged umbrella sampling simulations provide insight into migration barriers and ligand dissociation barriers and analysis of the trajectories yield additional insight into the structural dynamics of fundamental processes. Also, such simulations are useful tools to quantify relative stability changes for situations where experiments are difficult. This is illustrated for NO-bound myoglobin. For the dissociation of benzonitrile from lysozyme it is found that long umbrella sampling simulations are required to approximately converge the free energy profile. Then, however, the resulting differential free energy between the bound and unbound state is in good agreement with estimates from molecular mechanics with generalized Born surface area simulations. Furthermore, comparing the barrier height for ligand escape suggests that ligand dissociation contains a non-equilibrium component.     

Structural studies of mutants of the lysozyme of bacteriophage T4. The temperature-sensitive mutant protein Thr157----Ile

To understand the roles of individual amino acids in the folding and stability of globular proteins, a systematic structural analysis of mutants of the lysozyme of bacteriophage T4 has been undertaken. The isolation, characterization, crystallographic refinement and structural analysis of a temperature-sensitive lysozyme in which threonine 157 is replaced by isoleucine is reported here. This mutation reduces the temperature of the midpoint of the reversible thermal denaturation transition by 11 deg.C at pH 2.0. Electron density maps showing differences between the wild-type and mutant X-ray crystal structures have obvious features corresponding to the substitution of threonine 157 by isoleucine. There is little difference electron density in the remainder of the molecule, indicating that the structural changes are localized to the site of the mutation. High-resolution crystallographic refinement of the mutant lysozyme structure confirms that it is very similar to wild-type lysozyme. The largest conformational differences are in the gamma-carbon of residue 157 and in the side-chain of Asp159, which shift 1.0 A and 1.1 A, respectively. In the wild-type enzyme, the gamma-hydroxyl group of Thr157 participates in a network of hydrogen bonds. Substitution of Thr157 with an isoleucine disrupts this set of hydrogen bonds. A water molecule bound in the vicinity of Thr155 partially restores the hydrogen bond network in the mutant structure, but the buried main-chain amide of Asp159 is not near a hydrogen bond acceptor. This unsatisfied hydrogen-bonding potential is the most obvious reason for the reduction in stability of the temperature-sensitive mutant protein.     

Determination of the Backbone Mobility of Ribonuclease T1 and its 2′GMP Complex Using Molecular Dynamics Simulations and NMR Relaxation Data

Abstract

The results of 1-nanosecond molecular dynamics simulations of the enzyme ribonuclease T1 and its 2′GMP complex in water are presented. A classification of the angular reorientations of the backbone amide groups is achieved via a transformation of NH-vector trajectories into several coordinate frames, thus unravelling contributions of NH-bond librations and backbone dihedral angle fluctuations.

The former turned out to be similar for all amides, as characterized by correlation times of librational motions in a subpicosecond scale, angular amplitudes of about 10–12° for out-of-peptide-plane displacements of the NH-bond and 3–5° for the in-plane displacements, whereas the contributions of much slower backbone dihedral angle fluctuations strongly depend on the secondary structure. Correlation functions relevant for NMR were obtained and analyzed utilizing the ‘model-free’ approach (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546–4559,4559-4570; Clore et al., (1990) J. Am. Chem. Soc. 112, 4989–4991). The dependence of the amplitude of local motion on the residue location in the backbone is in good agreement with the results of NMR relaxation measurements and X-ray data. The protein dynamics is characterized by a highly restricted local motion of those parts of the backbone with defined secondary structure as well as by a high flexibility in loop regions. The comparison of results derived from different periods of the trajectory (of 50 ps and 1 ns duration, 1000 points sampled) reveals a dependence of the observed dynamic picture on the characteristic time scale of the experimental method used. Comparison of the MD data for the free and liganded enzyme clearly indicates a restriction of the mobility within certain regions of the backbone upon inhibitor binding.     

Molecular dynamics simulations and oxidation rates of methionine residues of granulocyte colony-stimulating factor at different pH values

To understand the connection between the conformation of a protein molecule and the oxidation of its methionine residues, we measured the rates of oxidation of methionine residues by H(2)O(2) in granulocyte colony-stimulating factor (G-CSF) as a function of pH and also studied the structural properties of this protein as a function of pH via molecular dynamics simulations. We found that each of the four methionine groups in G-CSF have significant and different rates of oxidation as a function of pH. Moreover, Met(1), in the unstructured N-terminal region, has a rate of oxidation as low as half that of free methionine. The structural properties of G-CSF as a function of pH are evaluated in terms of properties such as hydrogen bonding, deviations from X-ray structure, helical/helical packing, and the atomic covariance fluctuation matrix of alpha-carbons. We found that dynamics (structural fluctuations) are essential in explaining oxidation and that a static picture, such as that resulting from X-ray data, fails in this regard. Moreover, the simulation results also indicate that the solvent-accessible area, traditionally used to measure solvent accessibility of a protein site, of the sulfur atom of methionine residues does not correlate well with the rate of oxidation. Instead, we identified a structural property, average two-shell water coordination number, that correlates well with measured oxidation rates.     

Normal mode refinement: crystallographic refinement of protein dynamic structure. II. Application to human lysozyme.

The dynamic structure of a protein, human lysozyme, is determined by the normal mode refinement of X-ray crystal structure. This method uses the normal modes of both internal and external motions to distinguish the real internal dynamics from the external terms such as lattice disorder, and gives an anisotropic and concerted picture of atomic fluctuations. The refinement is carried out with diffraction data of 5.0 to 1.8 A resolution, which are collected on an imaging plate. The results of the refinement show: (1) Debye-Waller factor consists of two parts, highly anisotropic internal fluctuations and almost isotropic external terms. The former is smaller than the latter by a factor of 0.72 in the scale of B-factor. Therefore, the internal dynamics cannot be recognized directly from the apparent electron density distribution. (2) The internal fluctuations show basically similar features as those predicted by the normal mode analysis, with almost the same amplitude and a similar level of anisotropy. (3) Correlations of fluctuations are detected between two lobes forming the active site cleft, which move simultaneously in opposite directions. This corresponds to the hinge-bending motion of lysozyme.     

Relation between sequence and structure of HIV-1 protease inhibitor complexes: a model system for the analysis of protein flexibility.

The flexibility of different regions of HIV-1 protease was examined by using a database consisting of 73 X-ray structures that differ in terms of sequence, ligands or both. The root-mean-square differences of the backbone for the set of structures were shown to have the same variation with residue number as those obtained from molecular dynamics simulations, normal mode analyses and X-ray B-factors. This supports the idea that observed structural changes provide a measure of the inherent flexibility of the protein, although specific interactions between the protease and the ligand play a secondary role. The results suggest that the potential energy surface of the HIV-1 protease is characterized by many local minima with small energetic differences, some of which are sampled by the different X-ray structures of the HIV-1 protease complexes. Interdomain correlated motions were calculated from the structural fluctuations and the results were also in agreement with molecular dynamics simulations and normal mode analyses. Implications of the results for the drug-resistance engendered by mutations are discussed briefly.     

Partial Least-Squares Functional Mode Analysis: Application to the Membrane Proteins AQP1, Aqy1, and CLC-ec1

We introduce an approach based on the recently introduced functional mode analysis to identify collective modes of internal dynamics that maximally correlate to an external order parameter of functional interest. Input structural data can be either experimentally determined structure ensembles or simulated ensembles, such as molecular dynamics trajectories. Partial least-squares regression is shown to yield a robust solution to the multidimensional optimization problem, with a minimal and controllable risk of overfitting, as shown by extensive cross-validation. Several examples illustrate that the partial least-squares-based functional mode analysis successfully reveals the collective dynamics underlying the fluctuations in selected functional order parameters. Applications to T4 lysozyme, the Trp-cage, the aquaporin channels Aqy1 and hAQP1, and the CLC-ec1 chloride antiporter are presented in which the active site geometry, the hydrophobic solvent-accessible surface, channel gating dynamics, water permeability (p(f)), and a dihedral angle are defined as functional order parameters. The Aqy1 case reveals a gating mechanism that connects the inner channel gating residues with the protein surface, thereby providing an explanation of how the membrane may affect the channel. hAQP1 shows how the p(f) correlates with structural changes around the aromatic/arginine region of the pore. The CLC-ec1 application shows how local motions of the gating Glu(148) couple to a collective motion that affects ion affinity in the pore.