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Biomedical subjects

Charles L Brooks

Publications and source records attributed to Charles L Brooks.

At least 19 recordsLinked to original sources
PubMed2003

Generalized born model with a simple smoothing function.

Based on recent developments in generalized Born (GB) theory that employ rapid volume integration schemes (M. S. Lee, F. R. Salabury, Jr., and C. L. Brooks III, J Chem Phys 2002, 116, 10606) we have recast the calculation of the self-electrostatic solvation energy to utilize a simple smoothing function at the dielectric boundary. The present GB model is formulated in this manner to provide consistency with the Poisson-Boltzmann (PB) theory previously developed to yield numerically stable electrostatic solvation forces based on finite-difference methods (W. Im, D. Beglov, and B. Roux, Comp Phys Commun 1998, 111, 59). Our comparisons show that the present GB model is indeed an efficient and accurate approach to reproduce corresponding PB solvation energies and forces. With only two adjustable parameters--a(0) to modulate the Coulomb field term, and a(1) to include a correction term beyond Coulomb field--the PB solvation energies are reproduced within 1% error on average for a variety of proteins. Detailed analysis shows that the PB energy can be reproduced within 2% absolute error with a confidence of about 95%. In addition, the solvent-exposed surface area of a biomolecule, as commonly used in calculations of the nonpolar solvation energy, can be calculated accurately and efficiently using the simple smoothing function and the volume integration method. Our implicit solvent GB calculations are about 4.5 times slower than the corresponding vacuum calculations. Using the simple smoothing function makes the present GB model roughly three times faster than GB models, which attempt to mimic the Lee-Richards molecular volume.

Energy Transfer↗
PubMed2003

Temperature-dependent behavior of protein-chromophore interactions: a theoretical study of a blue fluorescent antibody.

The unusual temperature-dependent excited-state dynamics of a stilbene-antibody complex reported by Simeonov et al. are explored using theoretical methods. The anomalous temperature-dependent fluorescence emission and lifetime are shown to be the result of interplay among temperature-modulated protein flexibility, the excited-state potential surface for the stilbene central twist, and changes in the stilbene charge distribution upon excitation. Stilbene is found to possess a similar geometry and orientation within the antibody binding pocket at all temperatures in the ground state and at low temperatures (approximately 240 K) in the excited state. At higher temperatures (approximately 260 K), the excited-state conformation twists around the central double bond and adopts an alternate orientation within the binding pocket. These changes result from protein side chain and loop motions that are frozen out at lower temperatures and account for the observed red shift of the fluorescence emission spectrum (a calculated shift of 3.8 kcal mol-1 compares favorably with the approximately 5 kcal.mol-1 observed experimentally). Approximately 3.0 kcal mol-1 of this stabilization is global in nature and is not attributable to specific local interactions. Local interactions between stilbene and Tyr-B39 contribute approximately 0.8 kcal mol-1 to the fluorescence shift. The primary structural change in simulations of the high temperature excited state involves a decrease in the stilbene-tyrosine distance and a relative change in orientation of the aromatic rings. We identify several nearby charged residues that contribute to the fluorescence emission shift and provide targets for mutagenesis to probe the temperature-dependent dynamics of protein-chromophore interactions.

Light↗
PubMed2003

How dihydrofolate reductase facilitates protonation of dihydrofolate.

Dihydrofolate Reductase (DHFR) catalyzes the reduction of dihydrofolate (H2F) to tetrahydrofolate. On the basis of 10-12.5 ns molecular dynamics simulations of two conformations (closed and occluded) of the ternary DHFR/NADPH/H2F complex from Escherichia coli and a free energy perturbation approach, we have calculated the pKa value for the N5 atom in H2F. Our results suggest that the N5 atom in H2F is responsible for the pH dependency of the catalyzed reaction, meaning that DHFR facilitates protonation of H2F by approximately 4 pKa units. The mechanism behind this increase is due to favorable electrostatic interactions between the Asp27 residue and a proton at the N5 atom. The electrostatic interactions are enhanced by a hydrophobic active site, which to a large extent is made hydrophobic by the M20 loop in DHFR. Moreover, we find that the conformation imposed on H2F by DHFR to some extent also favors protonation of the N5 atom. Our results add support to previous findings and suggestions by Callender and co-workers [e.g., Deng, J.; Callender, R. J. Am. Chem. Soc. 1998, 120, 7730-7737] and explain why mutation of Asp27 may lead to severely reduced activity at neutral pH.

Catalysis↗
PubMed2003

Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy.

Combining structural data for the ribosome from x-ray crystallography and cryo-electron microscopy with dynamic models based on elastic network normal mode analysis, an atomically detailed picture of functionally important structural rearrangements that occur during translocation is elucidated. The dynamic model provides a near-atomic description of the ratchet-like rearrangement of the 70S ribosome seen in cryo-electron microscopy, and permits the identification of bridging interactions that either facilitate the conformational switching or maintain structural integrity of the 50S/30S interface. Motions of the tRNAs residing in the A and P sites also suggest the early stages of tRNA translocation as a result of this ratchet-like movement. Displacement of the L1 stalk, alternately closing and opening the intersubunit space near the E site, is observed in the dynamic model, in line with growing experimental evidence for the role of this structural component in facilitating the exiting of tRNA. Finally, a hinge-like transition in the 30S ribosomal subunit, similar to that observed in crystal structures of this complex, is also manifest as a dynamic mode of the ribosome. The coincidence of these dynamic transitions with the individual normal modes of the ribosome and the good correspondence between these motions and those observed in experiment suggest an underlying principle of nature to exploit the shape of molecular assemblies such as the ribosome to provide robustness to functionally important motions.

Binding Sites↗
PubMed2003

Correlated motion and the effect of distal mutations in dihydrofolate reductase.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002) Biochemistry 41, 12618-12628]. On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F/G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks.

Computer Simulation↗
PubMed2003

Identification of residues outside the two binding sites that are critical for activation of the lactogenic activity of human growth hormone.

Human growth hormone (hGH) binds lactogenic or somatotrophic receptors, creating active heterotrimeric complexes. Comparison of hGH structures, either free or bound to a single lactogenic or somatotrophic receptor, shows binding is associated with structural changes. Changes in hGH structure are unique when binding either lactogenic or somatotrophic receptors and they influence the spatial arrangement of residues constituting the second receptor-binding site. Using site-directed mutagenesis, we identified a contiguous set of largely hydrophobic residues that forms a motif communicating between the two receptor-binding sites of hGH. The residues are external to the receptor-binding epitopes and were identified when their mutation reduced site 2 function without changing site 1 function. The motif includes Phe44, Leu93, Tyr160, Leu163, and Tyr164, located in two hydrophobic clusters between the receptor-binding sites. Their mutation to Glu disrupts hydrophobic interactions and reduces lactogenic activity between 4.7- and 85-fold with little effect on somatotrophic activity or spectroscopic properties. These differential effects indicate that loss of lactogenic activity is not a result of global mis-folding. We propose the loss of lactogenic activity results from disruption of specific hydrophobic clusters that disables the site 1 binding-induced structuring of the second receptor-binding site.

Amino Acid Substitution↗
PubMed2003

The structural basis for biphasic kinetics in the folding of the WW domain from a formin-binding protein: lessons for protein design?

The mechanism of formation of beta-sheets is of great importance because of the significant role of such structures in the initiation and propagation of amyloid diseases. In this study we examine the folding of a series of three-stranded antiparallel beta-sheets known as WW domains. Whereas other WW domains have been shown to fold with single-exponential kinetics, the WW domain from murine formin-binding protein 28 has recently been shown to fold with biphasic kinetics. By using a combination of kinetics and thermodynamics to characterize a simple model for this protein, the origins of the biphasic kinetics is found to lie in the fact that most of the protein is able to fold without requiring one of the beta-hairpins to be correctly registered. The correct register of this hairpin is enforced by a surface-exposed hydrophobic contact, which is not present in other WW domains. This finding suggests the use of judiciously chosen surface-exposed hydrophobic pairs as a protein design strategy for enforcing the desired strand registry.

Amino Acid Sequence↗
PubMed2003

New analytic approximation to the standard molecular volume definition and its application to generalized Born calculations.

In a recent article (Lee, M. S.; Salsbury, F. R. Jr.; Brooks, C. L., III. J Chem Phys 2002, 116, 10606), we demonstrated that generalized Born (GB) theory provides a good approximation to Poisson electrostatic solvation energy calculations if one uses the same definitions of molecular volume for each. In this work, we present a new and improved analytic method for reproducing the Lee-Richards molecular volume, which is the most common volume definition for Poisson calculations. Overall, 1% errors are achieved for absolute solvation energies of a large set of proteins and relative solvation energies of protein conformations. We also introduce an accurate SASA approximation that uses the same machinery employed by our GB method and requires a small addition of computational cost. The combined methodology is shown to yield an efficient and accurate implicit solvent representation for simulations of biopolymers.

Algorithms↗
PubMed2003

Detailed analysis of grid-based molecular docking: A case study of CDOCKER-A CHARMm-based MD docking algorithm.

The influence of various factors on the accuracy of protein-ligand docking is examined. The factors investigated include the role of a grid representation of protein-ligand interactions, the initial ligand conformation and orientation, the sampling rate of the energy hyper-surface, and the final minimization. A representative docking method is used to study these factors, namely, CDOCKER, a molecular dynamics (MD) simulated-annealing-based algorithm. A major emphasis in these studies is to compare the relative performance and accuracy of various grid-based approximations to explicit all-atom force field calculations. In these docking studies, the protein is kept rigid while the ligands are treated as fully flexible and a final minimization step is used to refine the docked poses. A docking success rate of 74% is observed when an explicit all-atom representation of the protein (full force field) is used, while a lower accuracy of 66-76% is observed for grid-based methods. All docking experiments considered a 41-member protein-ligand validation set. A significant improvement in accuracy (76 vs. 66%) for the grid-based docking is achieved if the explicit all-atom force field is used in a final minimization step to refine the docking poses. Statistical analysis shows that even lower-accuracy grid-based energy representations can be effectively used when followed with full force field minimization. The results of these grid-based protocols are statistically indistinguishable from the detailed atomic dockings and provide up to a sixfold reduction in computation time. For the test case examined here, improving the docking accuracy did not necessarily enhance the ability to estimate binding affinities using the docked structures.

Algorithms↗
PubMed2002

A general method to quantify quasi-equivalence in icosahedral viruses.

A quantitative, atom-based, method is described for comparing protein subunit interfaces in icosahedral virus capsids with quasi-equivalent surface lattices. An integrated, normalized value (between 0 and 1) based on equivalent residue contacts (Q-score) is computed for every pair of subunit interactions and scores that are significantly above zero readily identify interfaces that are quasi-equivalent to each other. The method was applied to all quasi-equivalent capsid structures (T=3, 4, 7 and 13) in the Protein Data Bank and the Q-scores were interpreted in terms of their structural underpinnings. The analysis allowed classification of T=3 structures into three groups with architectures that resemble different polyhedra with icosahedral symmetry. The preference of subunits to form dimers in the T=4 human Hepatitis B virus capsid (HBV) was clearly reflected in high Q-scores of quasi-equivalent dimers. Interesting differences between the classical T=7 capsid and polyoma-like capsids were also identified. Application of the method to the outer-shell of the T=13 Blue tongue virus core (BTVC) highlighted the modest distortion between the interfaces of the general trimers and the strict trimers of VP7 subunits. Furthermore, the method identified the quasi 2-fold symmetry in the inner capsids of the BTV and reovirus cores. The results show that the Q-scores of various quasi-symmetries represent a "fingerprint" for a particular virus capsid architecture allowing particle classification into groups based on their underlying structural and geometric features.

Amino Acid Sequence↗
PubMed2002

Probing the folding free energy landscape of the Src-SH3 protein domain.

The mechanism and thermodynamics of folding of the Src homology 3 (SH3) protein domain are characterized at an atomic level through molecular dynamics with importance sampling. This methodology enables the construction of the folding free energy landscape of the protein as a function of representative reaction coordinates. We observe that folding proceeds in a downhill manner under native conditions, with early compaction and structure formation in the hydrophobic sheet consisting of the three central beta strands of the protein. This state bears considerable resemblance to the experimentally determined transition state for folding. Folding proceeds further with the formation of the second hydrophobic sheet consisting of the terminal strands and the RT loop. The final stages of folding appear to involve the formation of the hydrophobic core through the expulsion of water molecules bridging the two hydrophobic sheets. This work sheds new light on the complementary roles of sequence and topology in governing the folding mechanism of small proteins and provides further support for the role of water in facilitating the late stages in folding.

Animals↗
PubMed2002

Evaluating CASP4 predictions with physical energy functions.

Physical energy scoring functions based on implicit solvation models are tested by evaluating predictions from the most recent CASP4 competition. The best performing scoring functions are identified along with the best protocol for preparing structures before energies are evaluated. Ranking of structures with the best scoring functions is compared across CASP4 targets to establish when physical scoring functions can be expected to reliably distinguish structures that are most similar to the native fold in a set of misfolded or unfolded protein conformations. The results are used to interpret previous studies where scoring functions were tested on the standard decoy sets by Park, Levitt, and Baker. We show that the best physical scoring functions can be applied successfully in automated consensus scoring applications where a single best conformation has to be selected from a set of structures from different sources. Finally, the potential for better protein structure scoring functions is discussed with a suggestion for an empirically parameterized linear combination of energy components.

Animals↗
PubMed2002

Exploring global distortions of biological macromolecules and assemblies from low-resolution structural information and elastic network theory.

A theory of elastic normal modes is described for the exploration of global distortions of biological structures and their assemblies based upon low-resolution image data. Structural information at low resolution, e.g. from density maps measured by cryogenic electron microscopy (cryo-EM), is used to construct discrete multi-resolution models for the electron density using the techniques of vector quantization. The elastic normal modes computed based on these discretized low-resolution models are found to compare well with the normal modes obtained at atomic resolution. The quality of the normal modes describing global displacements of the molecular system is found to depend on the resolution of the synthetic EM data and the extent of reductionism in the discretized representation. However, models that reproduce the functional rearrangements of our test set of molecules are achieved for realistic values of experimental resolution. Thus large conformational changes as occur during the functioning of biological macromolecules and assemblies can be elucidated directly from low-resolution structural data through the application of elastic normal mode theory and vector quantization.

Adenylate Kinase↗
PubMed2002

The effects of ionic strength on protein stability: the cold shock protein family.

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.

Amino Acid Sequence↗
PubMed2002

The mechanism and pathway of pH induced swelling in cowpea chlorotic mottle virus.

Normal mode analysis based on a simplified energy function was used to study the swelling process of the icosahedral virus, cowpea chlorotic mottle virus (CCMV). Native state virus particles (coat proteins) of this T=3 icosahedral virus have been shown to undergo a large conformational change to a swollen state when metal ions are removed or the pH is raised. A normal mode analysis based on the native state capsid showed one preferential direction, a breathing mode, that explains the majority of the structural rearrangement necessary to bring the native structure close to the swollen state. From the native form of CCMV, the structure can be displaced along the direction of a single breathing mode by different amounts to create several candidate swollen structures and a putative pathway for virus expansion. The R-factor between these predicted swollen capsid structures and experimental electron density from cryoelectron microscopy (cryo-EM) measurements is then calculated to indicate how well each structure satisfies the experimental measurements on the swollen capsid state. A decrease of the crystallographic R-factor value from approximately 72% to approximately 49% was observed for these simple incremental displacements along the breathing mode. The simultaneous displacement of the native structure along other relevant (symmetric, non-degenerate) modes produce a structure with an R-factor of 45%, which is further reduced to 43.9% after minimization: a value in good accord with models based on the EM data at 28 A resolution. Based on the incrementally expanded structures, a pathway for the swelling process has been proposed. Analysis of the intermediate structures along this pathway indicates a significant loss of interactions at the quasi-3-fold interfaces occurs in the initial stages of the swelling process and this serves as a trigger for the compact to swollen transition. Furthermore, the pH dependent swelling appears to be triggered by the titration of a single residue with an anomalous pK(a) value in the unswollen particle.

Binding Sites↗
PubMed2002

Identifying native-like protein structures using physics-based potentials.

As the field of structural genomics matures, new methods will be required that can accurately and rapidly distinguish reliable structure predictions from those that are more dubious. We present a method based on the CHARMM gas phase implicit hydrogen force field in conjunction with a generalized Born implicit solvation term that allows one to make such discrimination. We begin by analyzing pairs of threaded structures from the EMBL database, and find that it is possible to identify the misfolded structures with over 90% accuracy. Further, we find that misfolded states are generally favored by the solvation term due to the mispairing of favorable intramolecular ionic contacts. We also examine 29 sets of 29 misfolded globin sequences from Levitt's "Decoys 'R' Us" database generated using a sequence homology-based method. Again, we find that discrimination is possible with approximately 90% accuracy. Also, even in these less distorted structures, mispairing of ionic contacts results in a more favorable solvation energy for misfolded states. This is also found to be the case for collapsed, partially folded conformations of CspA and protein G taken from folding free energy calculations. We also find that the inclusion of the generalized Born solvation term, in postprocess energy evaluation, improves the correlation between structural similarity and energy in the globin database. This significantly improves the reliability of the hypothesis that more energetically favorable structures are also more similar to the native conformation. Additionally, we examine seven extensive collections of misfolded structures created by Park and Levitt using a four-state reduced model also contained in the "Decoys 'R' Us" database. Results from these large databases confirm those obtained in the EMBL and misfolded globin databases concerning predictive accuracy, the energetic advantage of misfolded proteins regarding the solvation component, and the improved correlation between energy and structural similarity due to implicit solvation. Z-scores computed for these databases are improved by including the generalized Born implicit solvation term, and are found to be comparable to trained and knowledge-based scoring functions. Finally, we briefly explore the dynamic behavior of a misfolded protein relative to properly folded conformations. We demonstrate that the misfolded conformation diverges quickly from its initial structure while the properly folded states remain stable. Proteins in this study are shown to be more stable than their misfolded counterparts and readily identified based on energetic as well as dynamic criteria. In summary, we demonstrate the utility of physics-based force fields in identifying native-like conformations in a variety of preconstructed structural databases. The details of this discrimination are shown to be dependent on the construction of the structural database.

Algorithms↗