Ab initio molecular dynamics simulations of beta-D-glucose and beta-D-xylose degradation mechanisms in acidic aqueous solution

Ab initio molecular dynamics simulations were employed to investigate, with explicit solvent water molecules, beta-D-glucose and beta-D-xylose degradation mechanisms in acidic media. The rate-limiting step in sugar degradation was found to be protonation of the hydroxyl groups on the sugar ring. We found that the structure of water molecules plays a significant role in the acidic sugar degradation pathways. Firstly, a water molecule competes with the hydroxyl group on the sugar ring for protons. Secondly, water forms hydrogen bonds with the hydroxyl groups on the sugar rings, thus weakening the C-C and C-O bonds (each to a different degree). Note that the reaction pathways could be altered due to the change of relative stability of the C-C and C-O bonds. Thirdly, water molecules that are hydrogen-bonded to sugar hydroxyls could easily extract a proton from the reaction intermediate, terminating the reaction. Indeed, the sugar degradation pathway is complex due to multiple protonation probabilities and the surrounding water structure. Our experimental data support multiple sugar acidic degradation pathways.     

Deferiprone protects against doxorubicin-induced myocyte cytotoxicity

The iron chelating hydroxypyridinone deferiprone (CP20, L1) and the clinically approved cardioprotective agent dexrazoxane (ICRF-187) were examined for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle, both through reductive activation to its semiquinone form, and by the production of hydroxyl radicals mediated by its complex with iron. The results of this study showed that both deferiprone and dexrazoxane were able to protect myocytes from doxorubicin-induced lactate dehydrogenase release. Deferiprone quickly and efficiently removed iron(III) from its complex with doxorubicin. In addition, this study also showed that deferiprone rapidly entered myocytes and displaced iron from a fluorescence-quenched trapped intracellular iron-calcein complex, suggesting that in the myocyte, deferiprone should also be able to displace iron from its complex with doxorubicin. It was shown by electron paramagnetic resonance spectroscopy that under hypoxic conditions myocytes were able to reduce doxorubicin to its semiquinone free radical. Deferiprone also greatly reduced hydroxyl radical production by the iron(III)-doxorubicin complex in the xanthine oxidase/xanthine superoxide generating system. Together these results suggest that deferiprone may protect against doxorubicin-induced damage to myocytes by displacing iron bound to doxorubicin, or chelating free or loosely bound iron, thus preventing site-specific iron-based oxygen radical damage.     

4-Hydroxyphenylpyruvate dioxygenase: a hybrid density functional study of the catalytic reaction mechanism

Density functional calculations using the B3LYP functional has been used to study the reaction mechanism of 4-hydroxyphenylpyruvate dioxygenase. The first part of the catalytic reaction, dioxygen activation, is found to have the same mechanism as in alpha-ketoglutarate-dependent enzymes; the ternary enzyme-substrate-dioxygen complex is first decarboxylated to the iron(II)-peracid intermediate, followed by heterolytic cleavage of the O-O bond yielding an iron(IV)-oxo species. This highly reactive intermediate attacks the aromatic ring at the C1 position and forms a radical sigma complex, which can either form an arene oxide or undergo a C1-C2 side-chain migration. The arene oxide is found to have no catalytic relevance. The side-chain migration is a two-step process; the carbon-carbon bond cleavage first affords a biradical intermediate, followed by a decay of this species forming the new C-C bond. The ketone intermediate formed by a 1,2 shift of an acetic acid group rearomatizes either at the active site of the enzyme or in solution. The hypothetical oxidation of the aromatic ring at the C2 position was also studied to shed light on the 4-HPPD product specificity. In addition, the benzylic hydroxylation reaction, catalyzed by 4-hydroxymandelate synthase, was also studied. The results are in good agreement with the experimental findings.     

Theoretical studies of enzymic reactions: Dielectric,electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme

A general method for detailed study of enzymic reactions is presented. The method considers the complete enzyme-substrate complex together with the surrounding solvent and evaluates all the different quantum mechanical and classical energy factors that can affect the reaction pathway. These factors include the quantum mechanical energies associated with bond cleavage and charge redistribution of the substrate and the classical energies of steric and electrostatic interactions between the substrate and the enzyme. The electrostatic polarization of the enzyme atoms and the orientation of the dipoles of the surrounding water molecules is simulated by a microscopic dielectric model. The solvation energy resulting from this polarization is considerable and must be included in any realistic calculation of chemical reactions involving anything more than an isolated molecule in vacuo. Without it, acidic groups can never become ionized and the charge distribution on the substrate will not be reasonable. The same dielectric model can also be used to study the reaction of the substrate in solution. In this way the reaction in solution can be compared with the enzymic reaction.In this paper we study the stability of the carbonium ion intermediate formed in the cleavage of a glycosidic bond by lysozyme. It is found that electrostatic stabilization is an important factor in increasing the rate of the reaction step that leads to the formation of the carbonium ion intermediate. Steric factors, such as the strain of the substrate on binding to lysozyme, do not seem to contribute significantly.     

Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging

Organophosphorus poisons (OP) bind covalently to the active-site serine of cholinesterases. The inhibited enzyme can usually be reactivated with powerful nucleophiles such as oximes. However, the covalently bound OP can undergo a suicide reaction (termed aging) yielding nonreactivatable enzyme. In human butyrylcholinesterase (hBChE), aging involves the residues His438 and Glu197 that are proximal to the active-site serine (Ser198). The mechanism of aging is known in detail for the nerve gases soman, sarin, and tabun as well as the pesticide metabolite isomalathion. Aging of soman- and sarin-inhibited acetylcholinesterase occurs by C-O bond cleavage, whereas that of tabun- and isomalathion-inhibited acetylcholinesterase occurs by P-N and P-S bond cleavage, respectively. In this work, the crystal structures of hBChE inhibited by the ophthalmic reagents echothiophate (nonaged and aged) and diisopropylfluorophosphate (aged) were solved and refined to 2.1, 2.25, and 2.2 A resolution, respectively. No appreciable shift in the position of the catalytic triad histidine was observed between the aged and nonaged conjugates of hBChE. This absence of shift contrasts with the aged and nonaged crystal structures of Torpedo californica acetylcholinesterase inhibited by the nerve agent VX. The nonaged hBChE structure shows one water molecule interacting with Glu197 and the catalytic triad histidine (His438). Interestingly, this water molecule is ideally positioned to promote aging by two mechanisms: breaking either a C-O bond or a P-O bond. Pesticides and certain stereoisomers of nerve agents are expected to undergo aging by breaking the P-O bond.     

Oxidative destruction of DNA by the adriamycin-iron complex

The 2:1 adriamycin-Fe(III) complex is able to bind to DNA and to catalyze its oxidative destruction. The binding of the drug-metal complex to DNA is indicated by characteristic spectral changes which are different from those seen with adriamycin intercalation and by the propensity of the drug-metal complex to precipitate DNA. Furthermore, intercalated adriamycin appears not to be available for iron binding. The resulting ternary complex is quite stable: it is not disrupted by incubation in the presence of EDTA and can be isolated by using Sephadex G-50 column chromatography. Disruption of the ternary complex requires vigorous conditions (extraction with phenol at 60 degrees C). The adriamycin-iron complex in free solution has the capacity to catalyze the reduction of oxygen by thiols. The DNA-bound drug-metal complex preserves this capacity over a wide range of complex/DNA ratios. As a consequence of this thiol-dependent oxygen reduction, DNA is cleaved. This thiol-dependent DNA cleavage has been shown to require hydrogen peroxide as an intermediate product. These results have led us to propose that the thiol-dependent DNA cleavage reaction has two stages involving (1) reduction of oxygen leading to hydrogen peroxide and then (2) peroxide-dependent DNA cleavage. An unusual property of this reaction is that the cleavage is not random but gives rise to a defined 2300 base pair fragment.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

Effects of cholesterol side-chain groups and adrenodoxin binding on the vibrational modes of carbon monoxide bound to cytochrome P-450scc: implications of the productive and nonproductive substrate bindings.

Effects of the bindings of cholesterol and its hydroxylated analogues on the Fe-CO stretching and the C-O stretching vibrations of cytochrome P-450scc-CO complex were examined by resonance Raman and FT-IR spectroscopies to reveal the spatial relationship between the steroid side-chain groups and the heme-bound C-O moiety at the active center. These C-O and Fe-CO vibrations exhibited considerable variations depending on the steroids used; however, analyses on the nu Fe-CO vs nu C-O plot for cytochrome P-450scc indicated the absence of the negative correlation between these two vibrations, which is common among various Fe(2+)-porphyrin-CO complexes having imidazole ligands. Rather, we noticed the existence of two groups depending on substrates, the one exhibiting C-O infrared absorption bands in the region from 1930 to 1940 cm-1 and higher enzymatic turnover numbers in the reconstituted enzymatic systems and the other exhibiting C-O infrared absorption bands in the region above 1945 cm-1 and lower enzymatic turnover numbers. Thus, the former substrate group is likely to be fitted into the substrate binding site in the efficient "productive substrate binding" structure, whereas the latter group may be bound to the enzyme in the structure not suitable for the efficient enzymatic reaction ("nonproductive substrate binding" conformation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetate C-C bond formation and decomposition in the anaerobic world: the structure of a central enzyme and its key active-site metal cluster

The structure of carbon monoxide dehydrogenase/acetyl-coenzyme A synthase (CODH/ACS), a central enzyme in the anaerobic metabolism of acetyl-coenzyme A (acetyl-CoA), has been solved to a resolution of 2.2A. The active-site metal cluster responsible for catalyzing acetyl C-C bond synthesis and cleavage, designated the A center, was identified as an Fe(4)S(4) iron sulfur cluster with one of its cysteine thiolates acting as a bridge to an adjacent binuclear metal site. Nickel was found at one position in the binuclear site and the other metal was indicated to be copper - a surprising result, implying a previously unrecognized role for copper. Details of the A center provided new insight into the unusual organometallic mechanism of acetyl C-C bond formation and cleavage, with substantial conformational changes indicated for binding of the large methylcorrinoid protein substrate, and a unique intramolecular channel acting to contain carbon monoxide within the protein and transfer it to the site needed for acetyl-CoA synthesis.     

Influence of metal ions on the ribonuclease P reaction. Distinguishing substrate binding from catalysis.

A high yield, photoactivated cross-linking reaction between a modified tRNA and RNase P RNA was used as a quantitative assay of substrate binding affinity. The cross-linking assay allows the effects of metal ions on substrate binding to be measured independently and in the absence of the pre-tRNA cleavage reaction. The results of this assay, in conjunction with the conventional cleavage assay, support the following conclusions about the nature of the RNase P RNA-tRNA binding interaction. (i) Monovalent cations act primarily to enhance enzyme-substrate binding, presumably by functioning as counterions. This enhancement can be attributed to a reduction in the tRNA off-rate. (ii) Although divalent cation is required for cleavage, the enzyme-substrate complex can form in the absence of divalent cation; the essential role of divalent cation in the reaction is thus catalytic. (iii) Ca2+ is as efficient as Mg2+ in promoting binding but supports catalysis only at a low rate.     

Green and sustainable route for oxidative depolymerization of lignin: New platform for fine chemicals and fuels

Depolymerization of lignin biomass to its value-added chemicals and fuels is pivotal for achieving the goals for sustainable society, and therefore has acquired key interest among the researchers worldwide. A number of distinct approaches have evolved in literature for the deconstruction of lignin framework to its mixture of complex constituents in recent decades. Among the existing practices, special attention has been devoted for robust site selective chemical transformation in the complex structural frameworks of lignin. Despite the initial challenges over a period of time, oxidation and oxidative cleavage process of aromatic building blocks of lignin biomass toward the fine chemical synthesis and fuel generation has improved substantially. The development has improved in terms of cost effectiveness, milder reaction conditions, and purity of compound individuals. These aforementioned oxidative protocols mainly involve the breaking of C-C and C-O bonds of complex lignin frameworks. More precisely in the line with environmentally friendly greener approach, the catalytic oxidation/oxidative cleavage reactions have received wide spread interest for their mild and selective nature toward the lignin depolymerization. This mini-review aims to provide an overview of recent developments in the field of oxidative depolymerization of lignin under greener and environmentally benign conditions. Also, these oxidation protocols have been discussed in terms of scalability and recyclability as catalysts for different fields of applications.     

Combined QM/MM calculations of active-site vibrations in binding process of P450cam to putidaredoxin

Combined QM/MM calculations of the active-site of cytochrome P450cam have been performed before and after the binding of P450cam to putidaredoxin. The calculations were carried out for both a 5-coordinated and a 6-coordinated active-site of cytochrome P450cam, with either a water molecule or a carbon monoxide molecule as a 6th distal ligand. An experimentally observed increase in the Fe-S stretching frequency that occurs after cytochrome P450cam binds to putidaredoxin, has been reproduced in our study. Experimentally observed changes in the Fe-C and C-O vibration frequencies that occur after binding of both proteins, have also been reproduced in our study. The computed increase of the Fe-S and Fe-C stretching frequencies is correlated with a corresponding decrease of the Fe-S and Fe-C interatomic distances. According to our calculations, for the active-site with carbon monoxide in the triplet electronic state, the binding process increases the spin densities on the iron and sulfur atoms, which changes the Fe-C and C-O stretching frequencies in opposite directions, in agreement with experimental data.     

Studies on the nature of interaction of iron(III) with alginates

The interactions between the polysaccharide alginate and iron(III) were investigated. The solution properties were studied through pH-metry, viscometry, zeta potential and particle size measurements. In the presence of alginate, iron(III) was stabilized and no precipitation was observed. Studies indicate that iron(III)-alginate system was more stable than iron(III) or alginate alone. The binding constant is of the order of 10(4) M(-1). A case for 'site binding model' for the interaction between alginate and Fe(III) has been made based on the studies using circular dichroism and zeta potential experiments. The number of binding sites per molecule of alginate has been estimated to be 66. This indicates that the alginate can bind more number of Fe(III) ions and thus provide a stable complex which can find wide industrial applications.     

Two types of conformers with distinct Fe-C-O configuration in the ferrous CO complex of horseradish peroxidase. Resonance Raman and infarared spectroscopic studies with native and deuteroheme-substituted enzymes

The presence of at least two types of conformers in the ferrous CO complex of horseradish peroxidase has been demonstrated with the use of native and deuteroheme-substituted enzymes. Type I conformers, predominant in acidic pH, exhibited both an Fe-CO stretching and an Fe-C-O bending Raman line together with an infrared C-O stretch band below 1920 em-1. On the other hand, type II conformers, dominant species in alkaline pH, showed only an Fe-CO stretching Raman line with the C-O stretch above 1930 cm-1. They were interconvertible either by the changes in pH or by the binding of benzhydroxamate, a substrate for the enzyme. The pKa value for the pH-dependent interconversion of CO complex of deuteroheme-substituted enzyme was 8.3. These findings were interpreted to mean that the bound CO molecule in type I conformers was more tilted over the heme-plane than that in type II conformers. A steric hindrance by the bound substrate or the protonated form of a distal amino acid residue, presumably of histidine, is considered to be the cause for the isomerization. By summarizing present and previous data on the vibrational frequencies of heme-carbonyl complexes, we found that there are inverse-linear relationships between the square of Fe-CO and that of C-O stretching frequencies, while squares of Fe-CO stretching and Fe-C-O bending frequencies were linearly correlated with each other. Also found is that the dissociation rate constant of CO molecule from heme-carbonyl complexes is a linear function of the Fe-CO stretching frequency. The significance of these results is discussed.     

Reactions of ketones with aromatics in acid media. The effect of trifluoromethyl groups and the acidity media. A theoretical study

The reactions of acetone, 2,2,2-trifluoroacetone and hexafluoroacetone in methanesulfonic (MSA) and triflic acids (TFSA) with benzene have been studied at M06-2X/6-311+G(d,p) level using cluster-continuum model, where the carbonyl group is explicitly solvated by acid molecules. The introduction of a trifluoromethyl group into the ketone structure reduces the activation energy of the tetrahedral intermediates formation due to an increase of the electrophilicity of the carbonyl group and raises the activation and the reaction energies of the C-O bond cleavage in formed carbinol due to the destabilization of the corresponding carbocation. The introduction of the second trifluoromethyl group inhibits the hydroxyalkylation reaction due to a very strong increase of the reaction and activation energies of the C-O bond cleavage which becomes the rate determining step. The most important catalytic effect of TFSA compared to MSA is not the protonation of the ketone carbonyl, but the reduction of the activation and reaction energies of the carbinol C-O bond cleavage due to better protosolvation properties. Even for TFSA no complete proton transfer to carbonyl oxygen has been observed for free ketones. Therefore, the protonation energies of free ketones cannot be considered as a measure of ketone reactivity in the hydroxyalkylation reaction.

Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells

Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C-C phenol-coupling and intermolecular C-O phenol-coupling reactions. We report here the isolation and characterization of a C-C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C-O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C-C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C-C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.     

Single-turnover kinetics of homoprotocatechuate 2,3-dioxygenase

Homoprotocatechuate 2,3-dioxygenase isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the substrate aromatic ring. In contrast to other members of the ring cleaving dioxygenase family, the transient kinetics of the extradiol dioxygenase catalytic cycle have been difficult to study because the iron is nearly colorless and EPR silent. Here, it is shown that the reaction cycle kinetics can be monitored by utilizing the alternative substrate 4-nitrocatechol (4NC), which is also cleaved in the proximal extradiol position. Changes in the optical spectrum of 4NC occurring as a result of ionization, environmental changes, and ring cleavage allow both the substrate binding and product formation phases of the reaction to be studied. It is shown that substrate binding occurs in a four-step process probably involving binding to two ionization states of the enzyme at different rates. Following an initial rapid binding of the monoanionic 4NC in the active site, slower binding to the Fe(II) and conversion to the dianionic form occur. The bound dianionic 4NC reacts rapidly with O(2) in four additional steps, apparently occurring in sequence. On the basis of the optical properties of the intermediates, these steps are hypothesized to be O(2) binding to the iron, isomerization of the resulting complex, ring opening, and product release. The natural substrate appears to form the same intermediates but with much larger rate constants. These are the first transient intermediates to be reported for an extradiol dioxygenase reaction.     

Human topoisomerase I cleavage complexes are repaired by a p53-stimulated recombination-like reaction in vitro

Several studies have shown that human topoisomerase I (htopoI) cleaves in the vicinity of various DNA lesions and thereby forms covalent intermediates known as ‘cleavage complexes’. Such complexes are detrimental to cells if they are not repaired. Therefore, it is generally accepted that repair pathways must exist for such lesions. We have demonstrated that a htopoI cleavage complex can be recognized by a second topoisomerase I molecule and thereby perform a so-called htopoI ‘double cleavage’ in vitro. In addition, we found that the double cleavage reaction was stimulated by p53. Here we show that the double cleavage reaction results in the removal of the original htopoI cleavage complex and the generation of a single-stranded gap of ~13 nt. This gap supports a sequence-dependent DNA recombination reaction mediated by the second htopoI molecule. Furthermore, we show that p53 strongly stimulates the recombination reaction. We suggest that this reaction may represent a novel p53-dependent topoisomerase I-induced recombination repair (TIRR) pathway for htopoI cleavage complexes.     

Structure and mechanism of GDP-mannose glycosyl hydrolase, a Nudix enzyme that cleaves at carbon instead of phosphorus

GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to GDP and sugar by substitution with inversion at C1 of the sugar. The enzyme has a modified Nudix motif and requires one divalent cation for activity. The 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP complex, together with kinetic, mutational, and NMR data, suggests a mechanism for the GDPMH reaction. Several residues and the divalent cation strongly promote the departure of the GDP leaving group, supporting a dissociative mechanism. Comparison of the GDPMH structure with that of a typical Nudix hydrolase suggests how sequence changes result in the switch of catalytic activity from P-O bond cleavage to C-O bond cleavage. Changes in the Nudix motif result in loss of binding of at least one Mg(2+) ion, and shortening of a loop by 6 residues shifts the catalytic base by approximately 10 A.     

Partial cleavage mapping of the cytoskeletal protein vinculin. Antibody and talin binding sites.

Vinculin is a 1066-amino acid protein found at several types of actin-membrane junction. To locate sites of interest in the primary structure, a map was derived using partial cleavage reactions. Of several different types of cleavage tested, the most useful was the 5-5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) reaction which cuts at cysteine residues. About 30 well defined fragments were obtained from vinculin, and several methods were used to locate these products in the sequence. Comparison of the peptides generated from whole vinculin with those from the 90-kDa amino-terminal proteolytic fragment revealed which originated there. The use of [14C]cyanide in conjunction with DTNB showed which peptides contained the original amino terminus. Secondary cleavage with N-chlorosuccinimide, a tryptophan-specific reagent, helped locate fragments, although it led to apparent increases in molecular weight of the products. These experiments revealed the location of 10 of the major DTNB fragments on the sequence. This map was used to locate binding sites. The site of interaction between vinculin and the focal contact protein talin was mapped by binding labeled talin to the separated fragments. The binding site was found to be in the amino-terminal 325 amino acids. The binding site of a commercially obtained monoclonal antivinculin antibody was mapped using Western blotting of cleaved vinculin. It proved to bind in the central area of the molecule between amino acid residues 545 and 737. Thus the cysteine cleavage reaction products provide a map of general utility for locating features on the vinculin molecule.