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E W Knapp

Publications and source records attributed to E W Knapp.

18 recordsLinked to original sources
PubMed2001

How to guarantee optimal stability for most representative structures in the Protein Data Bank.

We proposed recently an optimization method to derive energy parameters for simplified models of protein folding. The method is based on the maximization of the thermodynamic average of the overlap between protein native structures and a Boltzmann ensemble of alternative structures. Such a condition enforces protein models whose ground states are most similar to the corresponding native states. We present here an extensive testing of the method for a simple residue-residue contact energy function and for alternative structures generated by threading. The optimized energy function guarantees high stability and a well-correlated energy landscape to most representative structures in the PDB database. Failures in the recognition of the native structure can be attributed to the neglect of interactions between different chains in oligomeric proteins or with cofactors. When these are taken into account, only very few X-ray structures are not recognized. Most of them are short inhibitors or fragments and one is a structure that presents serious inconsistencies. Finally, we discuss the reasons that make NMR structures more difficult to recognizeCopyright 2001 Wiley-Liss, Inc.

Computational Biology↗
PubMed2001

Factors determining the orientation of axially coordinated imidazoles in heme proteins.

Factors determining conformations of imidazole axially coordinated to heme in heme proteins were investigated by analyzing 693 hemes in 432 different crystal structures of heme proteins from the Protein Data Bank (PDB), where at least one histidine is ligated to heme. The results from a search of the PDB for protein structures were interpreted with molecular force field computations. Analysis of data from these crystal structures indicated that there are two main factors that determine the orientations of imidazole ligated to heme. These are the interactions of imidazole with the propionic acid side chains of heme and with the histidine backbone. From the analysis of the crystal structures of heme proteins, it turned out that the hydrogen bonding pattern is often not decisive, though it is probably used by nature to fine-tune the orientation of imidazole axially ligated to heme. We found that in many heme proteins the NdeltaH group of imidazole ligated to heme can assume a number of different hydrogen bonds and that in mutant structures the orientation of the ligated imidazole often does not change significantly, although the mutant altered the hydrogen bonding scheme involving the imidazole. Data from crystal structures of heme proteins show that there are preferred orientations of imidazoles with respect to heme. Generally, the NdeltaH group of imidazole is oriented toward the propionic acid groups of the heme. In some cases, the NdeltaH group of imidazole is close to only one of the propionic acid groups, but it is practically never oriented in the opposite direction. The imidazole also adopts a preferred orientation with respect to its histidine backbone such that the plane of the imidazole ring is practically never parallel to the Calpha-Cbeta bond of its histidine backbone. For a given conformation of histidine backbone with respect to heme, as well as imidazole with respect to histidine backbone, the orientation of the imidazole with respect to heme is uniquely determined, since the three orientations depend on each other. Hence, the interaction of the imidazole with the backbone also influences the orientation of the imidazole with respect to the heme. Force field computations are in agreement with experimental data. With this method, we showed that there is an energy minimum when the NdeltaH group of the imidazole is oriented toward the propionic acid groups and that there are minima of energy for orientations where the imidazole ring is orthogonal to the plane defined by the Calpha-Cbeta and Cbeta-Cgamma bonds of the histidine. The computations also demonstrated that these interactions are mainly of electrostatic origin. By taking into account these two major factors, we were able to understand the orientations of axially coordinated imidazoles for all groups of heme proteins, except for the group of cytochrome c peroxidase. In this group, the orientation of the imidazole is determined by a strong hydrogen bond of the NdeltaH group with Asp235.

Crystallography, X-Ray↗
PubMed2001

Artificial cytochrome b: computer modeling and evaluation of redox potentials.

We generated atomic coordinates of an artificial protein that was recently synthesized to model the central part of the native cytochrome b (Cb) subunit consisting of a four-helix bundle with two hemes. Since no X-ray structure is available, the structural elements of the artificial Cb were assembled from scratch using all known chemical and structural information available and avoiding strain as much as possible. Molecular dynamics (MD) simulations applied to this model protein exhibited root-mean-square deviations as small as those obtained from MD simulations starting with the crystal structure of the native Cb subunit. This demonstrates that the modeled structure of the artificial Cb is relatively rigid and strain-free. The model structure of the artificial Cb was used to determine the redox potentials of the two hemes by calculating the electrostatic energies from the solution of the linearized Poisson-Boltzmann equation (LPBE). The calculated redox potentials agree within 20 meV with the experimentally measured values. The dependence of the redox potentials of the hemes on the protein environment was analyzed. Accordingly, the total shift in the redox potentials is mainly due to the low dielectric medium of the protein, the protein backbone charges, and the salt bridges formed between the arginines and the propionic acid groups of the hemes. The difference in the shift of the redox potentials is due to the interactions with the hydrophilic side chains and the salt bridges formed with the propionic acids of the hemes. For comparison and to test the computational procedure, the redox potentials of the two hemes in the native Cb from the cytochrome bc(1) (Cbc(1)) complex were also calculated. Also in this case the computed redox potentials agree well with experiments.

Amino Acid Sequence↗
PubMed2001

The X-ray absorption spectroscopy Debye-Waller factors of an iron compound and of met-myoglobin as a function of temperature.

Protein dynamics can be characterized by the mean square displacements of the individual atoms of a molecule. This concept is extended to X-ray absorption spectroscopy (XAS) of proteins where the physical information in the Debye-Waller factor is in general neglected. In a first step, a procedure for the investigation of the temperature dependence of XAS spectra has been developed for a small iron compound. Subsequently, experiments have been performed on met-myoglobin. It is shown that the mean square displacements of XAS are smaller than those obtained by Mössbauer spectroscopy and far smaller than crystallographic mean square displacements. This behavior is explained by the different sensitivity of the methods. XAS measures a relative mean square displacement between the absorbing and backscattering atoms only. A comparison with mean square displacements calculated from normal modes shows that static displacements contribute significantly. It becomes obvious that the atoms of the active center show a high correlation of their motions.

Binding Sites↗
PubMed2001

Calculated pH-dependent population and protonation of carbon-monoxy-myoglobin conformers.

X-ray structures of carbonmonoxymyoglobin (MbCO) are available for different pH values. We used conventional electrostatic continuum methods to calculate the titration behavior of MbCO in the pH range from 3 to 7. For our calculations, we considered five different x-ray structures determined at pH values of 4, 5, and 6. We developed a Monte Carlo method to sample protonation states and conformations at the same time so that we could calculate the population of the considered MbCO structures at different pH values and the titration behavior of MbCO for an ensemble of conformers. To increase the sampling efficiency, we introduced parallel tempering in our Monte Carlo method. The calculated population probabilities show, as expected, that the x-ray structures determined at pH 4 are most populated at low pH, whereas the x-ray structure determined at pH 6 is most populated at high pH, and the population of the x-ray structures determined at pH 5 possesses a maximum at intermediate pH. The calculated titration behavior is in better agreement with experimental results compared to calculations using only a single conformation. The most striking feature of pH-dependent conformational changes in MbCO-the rotation of His-64 out of the CO binding pocket-is reproduced by our calculations and is correlated with a protonation of His-64, as proposed earlier.

Animals↗
PubMed2000

Metal ligand aromatic cation-pi interactions in metalloproteins: ligands coordinated to metal interact with aromatic residues.

Cation-pi interactions between aromatic residues and cationic amino groups in side chains and have been recognized as noncovalent bonding interactions relevant for molecular recognition and for stabilization and definition of the native structure of proteins. We propose a novel type of cation-pi interaction in metalloproteins; namely interaction between ligands coordinated to a metal cation--which gain positive charge from the metal--and aromatic groups in amino acid side chains. Investigation of crystal structures of metalloproteins in the Protein Data Bank (PDB) has revealed that there exist quite a number of metalloproteins in which aromatic rings of phenylalanine, tyrosine, and tryptophan are situated close to a metal center interacting with coordinated ligands. Among these ligands are amino acids such as asparagine, aspartate, glutamate, histidine, and threonine, but also water and substrates like ethanol. These interactions play a role in the stability and conformation of metalloproteins, and in some cases may also be directly involved in the mechanism of enzymatic reactions, which occur at the metal center. For the enzyme superoxide dismutase, we used quantum chemical computation to calculate that Trp163 has an interaction energy of 10.09 kcal mol(-1) with the ligands coordinated to iron.

Alcohol Dehydrogenase↗
PubMed2000

Electron transfer between the quinones in the photosynthetic reaction center and its coupling to conformational changes.

The electron transfer between the two quinones Q(A) and Q(B) in the bacterial photosynthetic reaction center (bRC) is coupled to a conformational rearrangement. Recently, the X-ray structures of the dark-adapted and light-exposed bRC from Rhodobacter sphaeroides were solved, and the conformational changes were characterized structurally. We computed the reaction free energy for the electron transfer from to Q(B) in the X-ray structures of the dark-adapted and light-exposed bRC from Rb. sphaeroides. The computation was done by applying an electrostatic model using the Poisson-Boltzmann equation and Monte Carlo sampling. We accounted for possible protonation changes of titratable groups upon electron transfer. According to our calculations, the reaction energy of the electron transfer from to Q(B) is +157 meV for the dark-adapted and -56 meV for the light-exposed X-ray structure; i.e., the electron transfer is energetically uphill for the dark-adapted structure and downhill for the light-exposed structure. A common interpretation of experimental results is that the electron transfer between and Q(B) is either gated or at least influenced by a conformational rearrangement: A conformation in which the electron transfer from to Q(B) is inactive, identified with the dark-adapted X-ray structure, changes into an electron-transfer active conformation, identified with the light-exposed X-ray structure. This interpretation agrees with our computational results if one assumes that the positive reaction energy for the dark-adapted X-ray structure effectively prevents the electron transfer. We found that the strongly coupled pair of titratable groups Glu-L212 and Asp-L213 binds about one proton in the dark-adapted X-ray structure, where the electron is mainly localized at Q(A), and about two protons in the light-exposed structure, where the electron is mainly localized at Q(B). This finding agrees with recent experimental and theoretical studies. We compare the present results for the bRC from Rb. sphaeroides to our recent studies on the bRC from Rhodopseudomonas viridis. We discuss possible mechanisms for the gated electron transfer from to Q(B) and relate them to theoretical and experimental results.

Crystallography, X-Ray↗
PubMed2000

A statistical mechanical method to optimize energy functions for protein folding.

We present a method for deriving energy functions for protein folding by maximizing the thermodynamic average of the overlap with the native state. The method has been tested by using the pairwise contact approximation of the energy function and generating alternative structures by threading sequences over a database of 1, 169 structures. With the derived energy function, most native structures: (i) have minimal energy and (ii) are thermodynamically rather stable, and (iii) the corresponding energy landscapes are smooth. Precisely, 92% of the 1,013 x-ray structures are stabilized. Most failures can be attributed to the neglect of interactions between chains forming polychain proteins and of interactions with cofactors. When these are considered, only nine cases remain unexplained. In contrast, 38% of NMR structures are not assigned properly.

Protein Conformation↗
PubMed2000

Structural alignment of ferredoxin and flavodoxin based on electrostatic potentials: implications for their interactions with photosystem I and ferredoxin-NADP reductase.

The two proteins ferredoxin and flavodoxin can replace each other in the photosynthetic electron transfer chain of cyanobacteria and algae. However, structure, size, and composition of ferredoxin and flavodoxin are completely different. Ferredoxin is a small iron-sulfur protein (approximately 100 amino acids), whereas flavodoxin is a flavin-containing protein (approximately 170 amino acids). The crystal structure of both proteins from the cyanobacteria Anabeana PCC 7120 is known. We used these two protein structures to investigate the structural basis of their functional equivalence. We apply the Hodgkin index to quantify the similarity of their electrostatic potentials. The technique has been applied successfully in indirect drug design for the alignment of small molecule and bioisosterism elucidation. It requires no predefined atom-atom correspondences. As is known from experiments, electrostatic interactions are most important for the association of ferredoxin and flavodoxin with their reaction partners photosystem I and ferredoxin-NADP reductase. Therefore, use of electrostatic potentials for the structural alignment is well justified. Our extensive search of the alignment space reveals two alignments with a high degree of similarity in the electrostatic potential. In both alignments, ferredoxin overlaps completely with flavodoxin. The active sites of ferredoxin and flavodoxin rather than their centers of mass coincide in both alignments. This is in agreement with electron microscopy investigations on photosystem I cross-linked to ferredoxin or flavodoxin. We identify residues that may have the same function in both proteins and relate our results to previous experimental data.

Algal Proteins↗
PubMed2000

Diffusion of water molecules in crystalline beta-cyclodextrin hydrates.

To understand the rapid diffusion mechanism of water molecules in the crystal lattice of hydrated beta-cyclodextrin (beta-CD), molecular dynamics (MD) simulations of crystalline beta-CD were performed at five different relative humidities corresponding to hydration states ranging from beta-CD-9.4H2O to beta-CD-12.3H2O, and in aqueous solution. The trajectories for the crystalline beta-CD hydrates had lengths of 4 ns each, whereas the simulation in aqueous solution extended to 2 ns. Transport of water molecules in the crystal was characterized in terms of a spatially varying diffusion constant and the main direction of diffusion, which were evaluated using newly developed algorithms. The main diffusion pathway winds through the cavities of adjacent doughnut shaped beta-CD molecules and is slightly slanted with respect to the crystallographic b-axis. Water molecules outside the beta-CD cavities have access to the main diffusion pathway. The diffusion constant for transport of water molecules along the main pathway calculated from the MD simulation data adopts 1/30 of the value in bulk water at room temperature. This is in agreement with estimates that can be made from experimental data on the adjustment of a beta-CD crystal to changes in relative humidity.

Algorithms↗
PubMed1998

Energetics of electron-transfer and protonation reactions of the quinones in the photosynthetic reaction center of Rhodopseudomonas viridis.

The electron-transfer reactions involving the quinones in the bacterial photosynthetic reaction center (bRC) are coupled to a proton uptake by the bRC. In this study, we calculated the energies of the different states of the bRC occurring during these electron-transfer and protonation reactions by an electrostatic model. We considered the possibility that titratable groups of the bRC can change their protonation during these reactions. The protonation probabilities of titratable groups were obtained by a Monte Carlo calculation. In contrast to earlier studies by other groups, we used atomic partial charges derived from quantum-chemical calculations. Our calculated reaction energies are in agreement with experiments. We found that the proton uptake by the bRC is coupled more strongly to changes of the redox state of the quinones than to changes of their protonation state. Thus, the proton uptake by the bRC occurs predominantly before the protonation of QB. According to our computations, the reduction of QB* - to the doubly negative state QB2- is energetically even more unfavorable in the bRC than in solution. Therefore, we suggest that the second electron transfer from QA to QB occurs after QB has received its first proton. We found that the QA. -QB. - state is more populated at pH 7.5 than the QA. -QB.H state. The low population of the QA. -QB.H state may be the reason why the singly protonated QB could not be detected spectroscopically. Our calculations imply that the first protonation of QB. - is a prerequisite for the second electron transfer between QA and QB. Therefore, a pH dependence of the equilibrium between the states QA. -QB. - and QA. -QB. H can also explain the experimentally observed pH dependence of the rate for the second electron-transfer step. On the basis of our calculated reaction energies, we propose the following sequence for the electron-transfer and protonation reactions: (1) first electron transfer from QA to QB, (2) first protonation of QB (at the distal oxygen close to Ser L223), (3) second electron transfer from QA to QB, and (4) second protonation of QB (at the proximal oxygen close to His L190).

Binding Sites↗
PubMed1997

Comparison of the physiologically equivalent proteins cytochrome c6 and plastocyanin on the basis of their electrostatic potentials. Tryptophan 63 in cytochrome c6 may be isofunctional with tyrosine 83 in plastocyanin.

The blue copper protein plastocyanin and the heme protein cytochrome c6 differ in composition and in structure but perform the same function in the photosynthetic electron-transport chain. We compare these two proteins on the basis of their electrostatic potentials in order to understand the structural basis of their functional equivalence. In the first approach, we use a monopole-dipole approximation of the electrostatic potentials to superimpose the proteins. The resulting alignment suggests that Tyr51 in cytochrome c6 corresponds to Tyr83 in plastocyanin. But since Tyr51 is not conserved in all known cytochrome c6 sequences, a physiological role of this residue is questionable. In a more sophisticated approach, we applied the recently-developed Fame (flexible alignment of molecule ensembles) algorithm, in which molecules are superimposed by optimizing the similarity of their electrostatic potentials with respect to the relative orientation of the molecules. On the basis of the Fame alignments of plastocyanin and cytochrome c6, we analyze the docking and the electron-transfer reactions of these two proteins with its physiological reaction partner cytochrome f. We derive functional analogies for individual amino acids in possible electron-transfer paths in the interprotein redox reactions. We identify two surface patches in cytochrome c6 that may be involved in electron-transfer paths. The hydrophobic patch with the exposed heme edge in cytochrome c6 may be equivalent to the hydrophobic patch with His87 in plastocyanin, whereas Trp63 in cytochrome c6 may be equivalent to Tyr83 in plastocyanin. An aromatic amino acid is present at the position of Trp63 in all known cytochrome c6 sequences. The electronic coupling between the heme and the copper site on the one side and several potentially important amino acid residues on the other is analyzed by the Pathways method. We have proposed recently that Lys65 of cytochrome f and Tyr83 of plastocyanin form a cation-pi system, which may be involved in a two-step mechanism of the electron-transfer reaction between these two proteins from higher plants. Now we corroborate this proposal by analyzing available amino acid sequences.

Algorithms↗
PubMed1996

Shift of the special pair redox potential: electrostatic energy computations of mutants of the reaction center from Rhodobacter sphaeroides.

Shifts of the special pair redox potential of the photosynthetic reaction center of Rhodobacter sphaeroides are considered for several point mutations [Lin. X., Murchison, H. A., Nagarijan, V., Parson, W. W., Allen, J. P., & Williams, J. C. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269] in the neighborhood of the special pair. The shifts are calculated from electrostatic energies by solving Poisson's equation for energy-minimized structures of the reaction center. Different conditions for the evaluation of the electrostatic energy are probed. To test the influence of the hydrogen bonding at the acetyl groups of the special pair, the orientation and torsion potential of the acetyl groups are varied. The calculated shifts of the midpoint potential of double and triple mutants can approximately be obtained from the corresponding shifts of the single point mutations. The calculated shifts agree with the measured values for all single and double mutants considered. However, a clear decision between different acetyl group conformations was only possible for the mutants HF(L168) and HF(L168) + LH(L131) where the calculated shifts of the redox potential agree with experiments only if the acetyl oxygen atom at DM points toward the Mg2+ ion of DL. This is corroborated by computations of the interaction energy of the acetyl group at DM, which adopts a lower value in the wild-type reaction center if its oxygen atom is bonded to the Mg2+ ion of DL.

Bacteriochlorophylls↗
PubMed1996

Structural fluctuations of myoglobin from normal-modes, Mössbauer, Raman, and absorption spectroscopy.

A normal-mode analysis of carbon monoxymyoglobin (MbCO) and deoxymyoglobin (Mb) with 170 water molecules is performed for (54)Fe and (57)Fe. A projection is defined that extracts iron out-of-plane vibrational modes and is used to calculate spectra that can be compared with those from resonance Raman scattering. The calculated spectra and the isotopic shift (57)Fe versus (54)Fe agree with the experimental data. At low temperatures the average mean square fluctuations (MSFs) of the protein backbone atoms agree with molecular dynamics simulation. Below 180 K the MSFs of the heme iron agree with the data from Mossbauer spectroscopy. The MSFs of the iron atom relative to the heme are an order of magnitude smaller than the total MSFs of the iron atom. They agree with the data from optical absorption spectroscopy. Thus the MSFs of the iron atom as measured by Mossbauer spectroscopy can be used to probe the overall motion of the heme within the protein matrix, whereas the Gaussian thermal line broadening of the Soret band and the resonance Raman bands can be used to detect local intramolecular iron-porphyrin motions.

Heme↗
PubMed1984

A consistent picture of protein dynamics.

Information about the protein dynamics of myoglobin obtained by x-ray and Mössbauer investigations is analyzed and compared with computer simulations. Computer simulations give correct amplitudes of mean-square displacements but fail in the description of the time dependence of motions. Our model describes protein dynamics at physiological temperatures as an overdamped diffusion-like motion in a restricted space. The fluctuations occur around the average conformation determined by x-ray structure analysis. The gain in entropy drives the molecule into the transition state and, in this way, accounts for its flexibility.

Computers↗