Introducing DInaMo: A Package for Calculating Protein Circular Dichroism Using Classical Electromagnetic Theory.

The dipole interaction model is a classical electromagnetic theory for calculating circular dichroism (CD) resulting from the π-π* transitions of amides. The theoretical model, pioneered by J. Applequist, is assembled into a package, DInaMo, written in Fortran allowing for treatment of proteins. DInaMo reads Protein Data Bank formatted files of structures generated by molecular mechanics or reconstructed secondary structures. Crystal structures cannot be used directly with DInaMo; they either need to be rebuilt with idealized bond angles and lengths, or they need to be energy minimized to adjust bond lengths and bond angles because it is common for crystal structure geometries to have slightly short bond lengths, and DInaMo is sensitive to this. DInaMo reduces all the amide chromophores to points with anisotropic polarizability and all nonchromophoric aliphatic atoms including hydrogens to points with isotropic polarizability; all other atoms are ignored. By determining the interactions among the chromophoric and nonchromophoric parts of the molecule using empirically derived polarizabilities, the rotational and dipole strengths are determined leading to the calculation of CD. Furthermore, ignoring hydrogens bound to methyl groups is initially explored and proves to be a good approximation. Theoretical calculations on 24 proteins agree with experiment showing bands with similar morphology and maxima.

Ionic strength dependence of F-actin and glycolytic enzyme associations: a Brownian dynamics simulations approach.

The association of glycolytic enzymes with F-actin is proposed to be one mechanism by which these enzymes are compartmentalized, and, as a result, may possibly play important roles for: regulation of the glycolytic pathway, potential substrate channeling, and increasing glycolytic flux. Historically, in vitro experiments have shown that many enzyme/actin interactions are dependent on ionic strength. Herein, Brownian dynamics (BD) examines how ionic strength impacts the energetics of the association of F-actin with the glycolytic enzymes: lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase (aldolase), and triose phosphate isomerase (TPI). The BD simulations are steered by electrostatics calculated by Poisson-Boltzmann theory. The BD results confirm experimental observations that the degree of association diminishes as ionic strength increases but also suggest that these interactions are significant, at physiological ionic strengths. Furthermore, BD agrees with experiments that muscle LDH, aldolase, and GAPDH interact significantly with F-actin whereas TPI does not. BD indicates similarities in binding regions for aldolase and LDH among the different species investigated. Furthermore, the residues responsible for salt bridge formation in stable complexes persist as ionic strength increases. This suggests the importance of the residues determined for these binary complexes and specificity of the interactions. That these interactions are conserved across species, and there appears to be a general trend among the enzymes, support the importance of these enzyme-F-actin interactions in creating initial complexes critical for compartmentation.

Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide whose biological activity has potential therapeutic value for many vascular related diseases. CGRP is a 37 amino acid neuropeptide that signals through a G protein-coupled receptor belonging to the secretin receptor family. Previous studies on the calcitonin-like receptor (CLR), which requires co-expression of the receptor-activity-modifying protein-1 (RAMP1) to function as a CGRP receptor, have shown an 18 amino acid N-terminus sequence important for binding CGRP. Moreover, several investigations have recognized the C-terminal amidated phenylalanine (F37) of CGRP as essential for docking to the mature receptor. Therefore, we hypothesize that hydrophobic amino acids within the previously characterized 18 amino acid CLR N-terminus domain are important binding contacts for the C-terminal phenylalaninamide of CGRP. RESULTS: Two leucine residues within this previously characterized CLR N-terminus domain, when mutated to alanine and expressed on HEK293T cells stably transfected with RAMP1, demonstrated a significantly decreased binding affinity for CGRP compared to wild type receptor. Additional decreases in binding affinity for CGRP were not found when both leucine mutations were expressed in the same CLR construct. Decreased binding characteristic of these leucine mutant receptors was observed for all CGRP ligands tested that contained the necessary amidated phenylalanine at their C-terminus. However, there was no difference in the potency of CGRP to increase cAMP production by these leucine mutant receptors when compared to wild type CLR, consistent with the notion that the neuropeptide C-terminal F37 is important for docking but not activation of the receptor. This observation was conserved when modified CGRP ligands lacking the amidated F37 demonstrated similar potencies to generate cAMP at both wild type and mutant CLRs. Furthermore, these modified CGRP ligands displayed a significant but similar loss of binding for all leucine mutant and wild type CLR because the important receptor contact on the neuropeptide was missing in all experimental situations. CONCLUSION: These results are consistent with previous structure-function investigations of the neuropeptide and are the first to propose specific CLR binding contacts for the amidated F37 of CGRP that are important for docking but not activation of the mature CGRP receptor.

BD SIMULATIONS OF THE IONIC STRENGTH DEPENDENCE OF THE INTERACTIONS BETWEEN TRIOSE PHOSPHATE ISOMERASE AND F-ACTIN.

Functional protein-protein interactions are essential for many physiological processes. For example, the association of glycolytic enzymes to F-actin is proposed to be one mechanism through which glycolytic enzymes are compartmentalized, and as a result, play essential roles such as regulation of the glycolytic pathway and increasing glycolytic flux. Many glycolytic enzymes including fructose-1,6-bisphophate aldolase, glyceraldedhye-3-phosphate dehydrogenase, and lactate dehydrogenase bind F-actin strongly. Other glycolytic enzymes including triose phosphate isomerase (TPI) do not interact with F-actin significantly. Herein, Brownian dynamics (BD) simulations determine the energetics of the association of F-actin with the glycolytic enzyme triose phosphate isomerase as a function of ionic strength. This is the first thorough control study examining how well BD reproduces the experimental observations that the binding of TPI to F-actin is very weak and falls off rapidly as ionic strength increases. The BD results confirm experimental observations that the degree of association diminishes as ionic strength increases and that the interaction of TPI with F-actin is weakly nonspecific to nonexistent.

Theoretical study of interactions between muscle aldolase and F-actin: insight into different species.

Interactions of the glycolytic enzyme, fructose-1,6-bisphosphate aldolase (aldolase), with F-actin may be one mechanism for the colocalization of glycolytic enzymes. Examination of these interactions in different animal species tests this hypothesis by observing whether binding sites are conserved across species. Brownian dynamics (BD) simulations provide descriptions of such protein-protein interactions with the muscle isoforms of zebra fish and human aldolase. The results are compared with previous results obtained for rabbit muscle and yeast. The aldolase binding groove previously determined in rabbit muscle is conserved in both the human and fish muscle isoforms. The nonspecific radial free energies of interaction are similar with fish being slightly weaker than human and rabbit: human, -2.27 +/- 0.05 kcal/mol; rabbit, -2.0 +/- 0.04 kcal/mol; and fish, -1.5 +/- 0.03 kcal/mol. BD results show a large Boltzmann population of complexes formed around the A/D and B/C grooves of aldolase with the most feasible binding mode comprising two aldolase subunits to subdomain I region of the actin subunits. These results show that the location of the important residues and binding site for fish and human aldolase is very similar to that in rabbit and that in different animals the binding site is conserved. This suggests that the binding interaction between aldolase and F-actin is general in animal muscles and is rendered possible and energetically favorable through the conservation of this binding site.

Highly diastereoselective synthesis of nucleoside adducts from the carcinogenic benzo[a]pyrene diol epoxide and a computational analysis.

A diastereoselective synthesis of the nucleoside adducts corresponding to a cis ring-opening of the carcinogen (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE-2) by 2'-deoxyadenosine and 2'-deoxyguanosine is described. The key intermediate (+/-)-10alpha-amino-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene was synthesized by a highly diastereoselective dihydroxylation wherein phenylboronic acid was a water surrogate. The resulting boronate ester was converted to a tetraol derivative in which two of the four hydroxyl groups (trans 7, 8) were protected as benzoate esters while the remaining two (cis 9, 10) were free. The cis glycol entity was then subjected to a reaction with 1-chlorocarbonyl-1-methylethylacetate to yield an intermediate chloro monoacetoxy dibenzoate. Displacement of the halide with azide, complete cleavage of the esters, and catalytic reduction of the azide yielded the requisite amino triol. Fluoride displacement from appropriately protected nucleoside derivatives, 6-fluoropurine 2'-deoxyribonucleoside and 2-fluoro-2'-deoxyinosine, by the amino triol then yielded diastereomeric pairs of diol epoxide-adducted 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) nucleosides. Small aliquots of these adducts were separated for characterization purposes. The present approach provides the first diastereoselective synthesis of the cis adducts of BaP DE-2 with 2'-deoxyguanosine as well as the first synthesis of both dA and dG adducts from a common intermediate. An informative analysis of the 1H NMR spectra of the cis adducts synthesized and comparisons to the trans adducts are reported. To gain insight into the diastereoselectivity in the key dihydroxylation step, a computational analysis, including molecular mechanics (MMFF94) and semiempirical AM1 geometry optimizations, yielded results that are in fairly good agreement with the experimental observations.

Theoretical UV circular dichroism of cyclo(L-Proline-L-Proline).

MP2, DFT, and molecular mechanics (AMBER, CVFF, and CFF91) geometry optimizations were performed on the cyclic dipeptide cyclo(L-Pro-L-Pro) starting from crystal structure data. Three stable conformations were identified as energy minima by all methods, but assignment of relative energy varied between the methods. The pi-pi transition feature of the UV circular dichroic (CD) spectrum was predicted for each minimized structure using the classical physics method of the dipole interaction model. The model was sensitive to the different conformations. The UV-CD predictions were compared individually and as a Boltzmann-weighted composite with published experimental CD spectra [Bowman, R. L.; Kellerman, M.; Johnson, W. C., Jr. Biopolymers 1983, 22, 1045]. For all structures, the original parameters of Applequist [Applequist, J. J. Chem. Phys. 1979, 71, 4324] with a bandwidth of 3000 cm(-1) most accurately replicated experiment, except for the CFF91 structures, which matched experiment best with a bandwidth of 4000 cm(-1). The inclusion of solvent by a continuum model did not significantly alter the minimized geometries obtained by molecular or quantum mechanics, but it did have an effect on the relative predicted energies of CFF91 and B3LYP conformations. The overall effect of solvent inclusion was negligible when Boltzmann-weighted spectra were considered. Gas-phase CFF91 structures were also reasonably good for prediction of CD spectra, and when water was included via a continuum model for energy calculations, the weighting scheme resembled that of the higher-level weightings. The CD calculated using the MP2/6-311G structures and energies for weighting were most descriptive of the 180 nm negative band in the experimental CD, but red-shifted the location of the 205 nm band. DFT structures were comparably, though not identically, as descriptive of the first pi-pi band, and did a better job with placement of the second (positive) pi-pi band. DFT calculations were less sensitive to basis set effect than the MP2 calculations, with 6-31G results in close agreement with 6-311G. The results suggest that it is possible to use geometries obtained from a variety of different methods (molecular mechanical or quantum mechanical) with the classical physics dipole interaction model to qualitatively reproduce the UV CD of model amides.

Glycolytic enzyme interactions with yeast and skeletal muscle F-actin.

Interaction of glycolytic enzymes with F-actin is suggested to be a mechanism for compartmentation of the glycolytic pathway. Earlier work demonstrates that muscle F-actin strongly binds glycolytic enzymes, allowing for the general conclusion that "actin binds enzymes", which may be a generalized phenomenon. By taking actin from a lower form, such as yeast, which is more deviant from muscle actin than other higher animal forms, the generality of glycolytic enzyme interactions with actin and the cytoskeleton can be tested and compared with higher eukaryotes, e.g., rabbit muscle. Cosedimentation of rabbit skeletal muscle and yeast F-actin with muscle fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by Scatchard analysis revealed a biphasic binding, indicating high- and low-affinity domains. Muscle aldolase and GAPDH showed low-affinity for binding yeast F-actin, presumably because of fewer acidic residues at the N-terminus of yeast actin; this difference in affinity is also seen in Brownian dynamics computer simulations. Yeast GAPDH and aldolase showed low-affinity binding to yeast actin, which suggests that actin-glycolytic enzyme interactions may also occur in yeast although with lower affinity than in higher eukaryotes. The cosedimentation results were supported by viscometry results that revealed significant cross-linking at lower concentrations of rabbit muscle enzymes than yeast enzymes. Brownian dynamics simulations of yeast and muscle aldolase and GAPDH with yeast and muscle actin compared the relative association free energy. Yeast aldolase did not specifically bind to either yeast or muscle actin. Yeast GAPDH did bind to yeast actin although with a much lower affinity than when binding muscle actin. The binding of yeast enzymes to yeast actin was much less site specific and showed much lower affinities than in the case with muscle enzymes and muscle actin.

Theoretical UV circular dichroism of aliphatic cyclic dipeptides.

Four cyclic dipeptides (piperazine-2,5-diones), cyclo(L-Pro-Gly), cyclo(L-Pro-L-Leu), cyclo(L-Ala-L-Ala), and cyclo(L-Pro-L-Ala), were modeled from crystal structure data. Conformations resulting from energy minimization using molecular mechanics were compared with traditional ab initio and density functional theory geometric optimizations for each dipeptide. In all computational cases, the gas phase was assumed. The pi-pi transition feature of the UV circular dichroic (CD) spectra was predicted for each peptide structure via the classical dipole interaction model. The dipole interaction model predicted CD spectra that qualitatively agreed with experiment when MP2 or DFT geometries were used. By coupling MP2 or DFT geometric optimizations with the classical physics method of the dipole interaction model, significantly better CD spectra were calculated than those using geometries obtained by molecular mechanics. Thus, one can couple quantum mechanical geometries with a classical physics model for calculation of circular dichroism.

Brownian dynamics of interactions between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mutants and F-actin.

Brownian dynamics simulations of computer models of GAPDH mutants interacting with F-actin emphasized the electrostatic nature of such interactions, and confirmed the importance of four previously identified lysine residues on the GAPDH structure in these interactions. Mutants were GAPDH models in which one or more of the previously identified lysines had been replaced with alanine. Simulations showed reduced binding of these mutants to F-actin compared to wild-type GAPDH. Binding was significantly reduced by mutating the four lysines; the specific electrostatic interaction energy of the quadruple mutant was -7.3 +/- 1.0 compared to -11.4 +/- 0.5 kcal/mol for the wild enzyme. The BD simulations also reaffirmed the importance of quaternary structure for GAPDH binding F-actin.

Brownian dynamics of interactions between aldolase mutants and F-actin.

Previous Brownian dynamics (BD) simulations (Ouporov IG, Knull HR and Thomasson KA 1999. Biophys. J. 76: 17-27) of complex formation between rabbit aldolase and F-actin have identified three lysine residues (K288, K293 and K341) on aldolase and acidic residues (DEDE) at the N-terminus of actin as important to binding. BD simulations of computer models of aldolase mutants with any of these lysine residues replaced by alanine show reduced binding energy; the greatest effect of a single substitution is for K341A, and replacement of all three lysines greatly reduces binding. BD simulations of wild-type rabbit aldolase vs altered F-actin show that binding is decreased if any one of the four N-terminal acidic residues is replaced by alanine and binding is greatly reduced if three or more of the N-terminal acidic residues are replaced; none of the four actin residues appear more critical for binding than the others.