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PubMed2002

Finance issue brief: direct access: year end report-2002.

Consumer demand for a less restrictive referral system has urged legislators, as well as the managed care industry and those in the medical field, to seek the best solution to the problem. The point of service (POS) plan was an early attempt by managed care plans to provide more freedom for enrollees. But POS plans are not always available or cost effective. Several of the larger managed care organizations, in an effort to improve enrollee and provider satisfaction, have eliminated or streamlined their referral processes. The American Medical Association has recommended that primary care and other medical specialty organizations collaborate in developing guidelines for specialty referrals, urging health plans to allow direct access in network without financial penalty as long as the access conforms with the established guidelines. In the meantime, legislators have responded to constituents by enacting measures that mandate direct access for specific types of care or standing referrals for ongoing treatment.

Chronic Disease↗

PubMed2003

Finance issue brief: direct access: year end report-2003.

Gatekeeping↗

PubMed2003

An open conformation of mammalian cytochrome P450 2B4 at 1.6-A resolution.

The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-A crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the alpha-helical and beta-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B' to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B' to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetry-related molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress.

Animals↗

PubMed2001

Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors.

Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix-turn-helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

Amino Acid Sequence↗

PubMed2002

Active rhodanese lacking nonessential sulfhydryl groups contains an unstable C-terminal domain and can be bound, inactivated, and reactivated by GroEL.

Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.

Amino Acid Sequence↗

PubMed1988

Conformational analysis of morphiceptin by NMR spectroscopy.

Three exorphins, beta-casomorphin-5, morphiceptin and its D-Pro4 analog, were studied in DMSO by means of 1H and 13C NMR spectroscopy, with the aim of detecting conformational features of potential biological significance for the mu opioid activity since the presence of two Pro residues restricts the accessible conformational space more than in all other peptides. It is found that the conformational mixtures present in solution contain relevant fractions of folded conformers, a feature that assures the observation of four different Tyr OH signals in the 500 MHz spectrum of morphiceptin. The conformer distribution of (very active) (D-Pro4)-morphiceptin is different from those of its (less active) congeners.

Endorphins↗

PubMed2000

Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis. A fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis.

LmrA, a membrane protein of Lactococcus lactis, extrudes amphiphilic compounds from the inner leaflet of the cytoplasmic membrane, using energy derived from ATP hydrolysis. A combination of total reflection Fourier transform infrared spectroscopy, (2)H/H exchange, and fluorescence quenching experiments was used to investigate the effect of nucleotide binding and/or hydrolysis on the structure of LmrA reconstituted into proteoliposomes. These measurements allowed us to describe secondary structure changes of LmrA during the catalytic cycle. The structure of LmrA is enriched in beta-sheet after ATP binding, and the protein recovers its initial secondary structure after ATP hydrolysis, when P(i) has been released. (2)H/H exchange and fluorescence quenching studies indicate that the protein undergoes two distinct tertiary structure changes during the hydrolysis process. Indeed, the protein alone is poorly accessible to the aqueous medium but adopts a more accessible conformation when ATP hydrolysis takes place. After ATP hydrolysis, but when P(i) is still associated with the protein, the accessibility is intermediate between these two states.

ATP-Binding Cassette Transporters↗

PubMed1991

Influence of environment on the antifolate drug trimethoprim: energy minimization studies.

Environmental effects on trimethoprim (TMP), an inhibitor of bacterial dihydrofolate reductase (DHFR), were investigated with energy minimizations in vacuo, in the crystal, and in aqueous solution. The conformations, harmonic dynamics, and energetics of the antibacterial drug calculated in these environments were compared with each other and with those of two enzyme-bound drugs. Valence and torsion angles and their energies and overall intra- and intermolecular energies compensated one another in the minimized TMP structures. The conformations of the isolated and aqueous molecules were similar to that of TMP bound to chicken liver DHFR, while the structures from the TMP crystal and from the Escherichia coli DHFR complex were unique. Since neither the small-molecule crystal nor a local minimum of the isolated molecule gave the conformation of TMP bound to the bacterial enzyme, a combination of several experimental and theoretical techniques may be necessary to probe accessible conformations of a molecule.

Animals↗

PubMed1999

Conformational and configurational features of acidic polysaccharides and their interactions with calcium ions: a molecular modeling investigation.

Modeling simulations have been performed on the four regular glycuronans: alpha-D-(1--->4) polygalacturonic, alpha-L-(1--->4) polyguluronic, beta-D-(1--->4) polymannuronic, and beta-D-(1--->4) polyglucuronic acids. The goal of this study was to characterize the similarities and differences in conformational and configurational behavior as well as in calcium binding in order to progress in the understanding of the physicochemical properties of the parent polysaccharides of industrial interest, namely pectin, alginate and glucuronan. This required the evaluation of the accessible conformational space for the disaccharide subunits of the four homopolymers, using the flexible residue protocol of the MM3 molecular mechanics procedure. The results were used to access the configurational statistics of representative polysaccharide chains, as well as for the determination of the regular polysaccharide helices and their conformational transitions. The surfaces of all regular helices likely to occur for each polyuronide were explored for cation binding using the GRID procedure. Both alpha-D-(1--->4) polygalacturonate and alpha-L-(1--->4) polyguluronate chains exhibit a high specificity for calcium binding, and have well-defined chelation sites. In contrast, beta-D-(1--->4) polymannuronate and beta-D-(1--->4) polyglucuronate chains do not display any stereospecificity for calcium binding. The results gathered from molecular modeling lead to a clear understanding of the different structural features that are displayed by the four ionic polymers.

Alginates↗

PubMed1996

Solution conformations of pectin polysaccharides: determination of chain characteristics by small angle neutron scattering, viscometry, and molecular modeling.

The solution behavior of pectin polysaccharides has been investigated by small angle neutron scattering (SANS), viscosimetric, and molecular modeling studies. The samples used in the experimental study were obtained from apple and citrus and had degrees of methylation ranging from 28 to 73%, with a rhamnose content lying between 0.6 and 2.2%. Persistence lengths, derived from intrinsic viscosity measurements, ranged from 59 to 126 A, whereas those derived by SANS were between 45 and 75 A. These values correspond to 10-17 monomer units. The modeling simulations were performed for both homogalacturonan itself and homogalacturonan carrying various degrees of rhamnose inserts (rhamnogalacturonan). This required the evaluation of the accessible conformational space for the eight disaccharides that represent the constituent repeating segments of the homogalacturonan and rhamnogalacturonan polysaccharides. For each dimer, complete conformational analysis was accomplished using the flexible residue method of the MM3 molecular mechanics procedure and the results used to access the configurational statistics of representative pectic polysaccharide chains. For homogalacturonan, an extended chain conformation having a persistence length of 135 A (corresponding to 30 monomers) was predicted. The inclusion of varying amounts of rhamnose units (5-25%) in the model in strict alternating sequence with galacturonate residues (equivalent to the rhamnogalacturonan "hairy region" chains) only slightly reduced the calculated persistence length. The extended overall chain conformation remained relatively unchanged as a consequence of the self-cancellation of the kinking effects of successive paired rhamnose units.

Carbohydrate Conformation↗

PubMed2005

Synergy of intramolecular hydrogen-bonding network in myo-inositol 2-monophosphate: theoretical investigations into the electronic structure, proton transfer, and pKa.

This work demonstrates the pivotal role that an intramolecular hydrogen-bonding network (intra-HBN) plays in the determination of the conformation of myo-inositol 2-monophosphate (Ins(2)P1), a member of the inositol phosphate family of compounds, which are important participants in the role that phosphates play in biological and environmental chemistry. For biologically significant compounds that contain phosphate and hydroxyl groups, Ins(2)P1 is a model system for studying both the primary forces that determine their conformations and their chemical properties from the effect of phosphate group addition. We performed ab initio calculations to determine the intra-HBN within important thermally accessible conformations for neutral Ins(2)P1 and its anions, Ins(2)P1(1-) and Ins(2)P1(2-). The results show that the global minima prefer 1a/5e structures where the phosphate group is in the axial position with all -OH groups in the equatorial positions. The calculations of transition state structures for ring inversion at each ionization state predict an activation energy of 18.16 kcal/mol for the neutral species in water, while the activation energy is lower for the charged compounds, 15.62 kcal/mol for Ins(2)P1(1-) and 12.48 kcal/mol for Ins(2)P1(2-). The pK(a) values of Ins(2)P1 were calculated by modeling the solvent as a polarizable continuum medium (PCM) and as explicit solvent molecules. These values are in good agreement with experimental data. A novel four-center pattern of hydrogen bonding was found to stabilize the system. The intramolecular proton transfer across a low barrier hydrogen bond between the charged phosphate and hydroxyl groups was found to occur under standard conditions with an activation energy that is less than 0.5 kcal/mol.

Electrons↗

PubMed1983

CNDO/2 molecular orbital calculations on the antifolate DAMP and some related species: structural geometries, ring distortions, change distributions and conformational characteristics.

Geometry-optimized CNDO/2 molecular orbital calculations were carried out on 2, 4-diamino-5-(1-adamantyl 1)-6-methyl pyrimidine (DAMP), a potent inhibitor of mammalian dihydrofolate reductase which is now in clinical trials, and on its inactive 5-(1-naphthyl) analogue (DNMP-1). Crystallographic data show that DAMP (as the ethylsulfonate salt) has a severely distorted, N1 protonated, pyrimidine ring and has steric crowding of the 6-methyl and adamantyl hydrogens whereas DNMP-2 (as a methanol complex) has a planar, nonprotonated pyrimidine ring that is nearly perpendicular to the naphthalene ring. The CNDO/2 results largely reproduce the crystal structure geometry and show that the ring distortions in DAMP are initiated by steric conflicts between the adamantyl group and the 4- and 6-substituents on the ring. In DNMP-1, the non-interfering naphthyl ring induces little strain within the pyrimidine ring and the effect of protonation is negligible. Rotation about the bond joining the two ring groups is restricted in DAMP by a broad barrier of ca. 8.0 kcal mol-1, and no conformation was successful in relieving steric conflicts and hence reducing the ring distortions. In DNMP-1, rotation is less hindered overall with a broad region of accessible conformational space and a maximum barrier of ca. 7.2 kcal mol-1 for the coplanar conformation. The electronic charge distributions of DAMP and DNMP-1 are almost identical and protonation is preferred at N1 rather than at N3 by ca. 3.7 kcal mol-1 for both DAMP and DNMP-1. The calculations establish that the present methodology can be useful as a predictive tool with regard to the structure and conformational characteristics of these and related species.

Adamantane↗

PubMed1989

Chemical design of cilazapril.

1. The three dimensional requirements for inhibition of ACE (angiotensin converting enzyme) were investigated in order to facilitate design of a more potent and selective antihypertensive agent. 2. All compounds designed possessed a bicyclic unit incorporating carboxylate and amidic carbonyl groups together with a thiol-bearing side chain. 3. NMR spectroscopy of the bicyclic units and molecular mechanics calculations enabled the possible positions of the thiol group to be studied. 4. Determination of the positions of the thiol group conferring best inhibition in the active site of ACE permitted the probable location of the active site zinc ion to be identified. The intention was to replace the thiol side chain with a homophenylalanine unit to bind to the zinc ion and also to occupy the S1 site which fits the Phe8 side chain of angiotensin I. 5. Examination of a torsional angle psi in a compound possessing poor inhibitory potency indicated correspondence to a high energy conformation of alanylproline. The bicyclic unit was modified to incorporate a seven-instead of a six-membered ring to bring psi into the range of an accessible conformation of alanylproline. The corresponding IC50 resulting indicated that psi was closer to that of the active conformations of enalaprilat and captopril. 6. Removal of one carbonyl improved the ACE inhibitory potency further. 7. The postulated active conformation of cilazaprilat is presented.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2006

Benchmarking pK(a) prediction.

BACKGROUND: pKa values are a measure of the protonation of ionizable groups in proteins. Ionizable groups are involved in intra-protein, protein-solvent and protein-ligand interactions as well as solubility, protein folding and catalytic activity. The pKa shift of a group from its intrinsic value is determined by the perturbation of the residue by the environment and can be calculated from three-dimensional structural data. RESULTS: Here we use a large dataset of experimentally-determined pKas to analyse the performance of different prediction techniques. Our work provides a benchmark of available software implementations: MCCE, MEAD, PROPKA and UHBD. Combinatorial and regression analysis is also used in an attempt to find a consensus approach towards pKa prediction. The tendency of individual programs to over- or underpredict the pKa value is related to the underlying methodology of the individual programs. CONCLUSION: Overall, PROPKA is more accurate than the other three programs. Key to developing accurate predictive software will be a complete sampling of conformations accessible to protein structures.

Catalysis↗

PubMed2003

Conformational and SAR analysis of NAALADase and PSMA inhibitors.

Prostate specific membrane antigen (PSMA) is a 110 kDa type II transmembrane protein that is expressed exclusively by prostate tumor cells and as such is a clear cellular target in the development of a new method for fast and reliable diagnosis of prostate cancer. PSMA is highly homologous to the neuropeptidase NAALADase, and it has been shown that inhibitors of NAALADase also strongly bind to PSMA. In an effort to better understand the structural basis of the inhibitory activity of more than 60NAALADase inhibitors synthesized and tested by our group, we used Monte Carlo calculations employing the Merck Molecular Force Field to explore the conformational space available to a set of PSMA inhibitors. Conformational analysis indicated that the lower the number of unique conformations accessible by an inhibitor, the greater the biological activity displayed by the compound against LnCAP cells. This suggests that the difference in activity is largely entropy based. The key conformations associated with high activity are used to develop a simple pharmacophore model that led to the design of new, conformationally restricted analogues with potentially high activity in rational drug design.

Antineoplastic Agents↗

PubMed1976

Tertiary structures of gastrin-like tetrapeptides.

Tertiary structures of gastrin-like tetrapeptide Trp-Met-Asp-Phe-NH2 and those substituted by Leu, Val or Gly for Met are studied. The lowest energy conformations of the side chains when the back bone is fixed in alpha-helix are obtained by modified minimization algorithm. It is suggested that protein folding proceeds in the accessible conformation space as a self-organization process leading to minimum energy conformation in this space.

Aspartic Acid↗

PubMed2003

Cooperative fluctuations of unliganded and substrate-bound HIV-1 protease: a structure-based analysis on a variety of conformations from crystallography and molecular dynamics simulations.

The dynamics of HIV-1 protease, both in unliganded and substrate-bound forms have been analyzed by using an analytical method, Gaussian network model (GNM). The method is applied to different conformations accessible to the protein backbone in the native state, observed in crystal structures and snapshots from fully atomistic molecular dynamics (MD) simulation trajectories. The modes of motion obtained from GNM on different conformations of HIV-1 protease are conserved throughout the MD simulations. The flaps and 40's loop of the unliganded HIV-1 protease structure are identified as the most mobile regions. However, in the liganded structure these flaps lose mobility, and terminal regions of the monomers become more flexible. Analysis of the fast modes shows that residues important for stability are in the same regions of all the structures examined. Among these, Gly86 appears to be a key residue for stability. The contribution of residues in the active site region and flaps to the stability is more pronounced in the substrate-bound form than in the unliganded form. The convergence of modes in GNM to similar regions of HIV-1 protease, regardless of the conformation of the protein, supports the robustness of GNM as a potentially useful and predictive tool.

Binding Sites↗

PubMed1987

The influence of sidechains on the calculated dimensions of three related bacterial polysaccharides.

The effect of van der Waals interactions between sidechain and backbone on the shape of three bacterial polysaccharides in solution has been investigated. The three polymers, namely, gellan, welan, and rhamsan, share the same four-sugar backbone repeating-unit. Gellan is unbranched, whereas welan and rhamsan display comblike branching. Consequently, the effect of chain branching on backbone conformation may be investigated. Van der Waals repulsive interactions of sidechain and backbone serve to limit, somewhat, the range of conformational freedom of the welan backbone in comparison to that of gellan. Attractive side-chain-backbone interactions, which may be as significant as 2-3 kcal/mol, predominate over much of the accessible conformational space of the welan backbone. Despite the strength of these interactions, the unperturbed shape of welan in solution is calculated to be very similar to that of the unbranched gellan. Attractive sidechain-backbone interactions in rhamsan have a modest influence on the conformational characteristics of the rhamsan backbone. The calculated, unperturbed conformation in solution is slightly more extended than that of gellan and welan, but the fundamental shape of the chain is changed only slightly. Significant differences in the physical properties of these polymers seem not to arise from differences in their random-coil conformations provoked by van der Waals interactions of sidechain and backbone. Other contributions to the sidechain-backbone interaction, e.g., hydrogen bonding, could be involved; interchain interactions are also likely to be important.

Carbohydrate Conformation↗