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PubMed1999

NMR solution structure of alpha-conotoxin ImI and comparison to other conotoxins specific for neuronal nicotinic acetylcholine receptors.

Alpha-Conotoxins, peptides produced by predatory species of Conus marine snails, are potent antagonists of nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels involved in synaptic transmission. We determined the NMR solution structure of the smallest known alpha-conotoxin, ImI, a 12 amino acid peptide that binds specifically to neuronal alpha7-containing nAChRs in mammals. Calculation of the structure was based on a total of 80 upper distance constraints and 31 dihedral angle constraints resulting in 20 representative conformers with an average pairwise rmsd of 0.44 A from the mean structure for the backbone atoms N, Calpha, and C' of residues 2-11. The structure of ImI is characterized by two compact loops, defined by two disulfide bridges, which form distinct subdomains separated by a deep cleft. Two short 310-helical regions in the first loop are followed by a C-terminal beta-turn in the second. The two disulfide bridges and Ala 9 form a rigid hydrophobic core, orienting the other amino acid side chains toward the surface. Comparison of the three-dimensional structure of ImI to those of the larger, 16 amino acid alpha-conotoxins PnIA, PnIB, MII, and EpI-also specific for neuronal nAChRs-reveals remarkable similarity in local backbone conformations and relative solvent-accessible surface areas. The core scaffold is conserved in all five conotoxins, whereas the residues in solvent-exposed positions are highly variable. The second helical region, and the specific amino acids that the helix exposes to solvent, may be particularly important for binding and selectivity. This comparative analysis provides a three-dimensional structural basis for interpretation of mutagenesis data and structure-activity relationships for ImI as well other neuronal alpha-conotoxins.

Amino Acid Sequence↗

PubMed1999

Phe310 in transmembrane VI of the alpha1B-adrenergic receptor is a key switch residue involved in activation and catecholamine ring aromatic bonding.

Pharmacophore mapping of adrenergic receptors indicates that the phenyl ring of catecholamine agonists is involved in receptor binding and activation. Here we evaluated Phe310, Phe311, and Phe303 in transmembrane VI (TMVI), as well as Tyr348 in TMVII of the alpha1B-adrenergic receptor (alpha1B-AR), which have been implicated in a catechol-ring interaction. Neither catecholamine docking studies nor mutagenesis studies of Phe311, Phe303, or Tyr348 supported a role for these residues in catechol-ring binding. By contrast, docking studies indicated that the Phe310 side chain is well positioned to interact with the catechol-ring, and substituted cysteine accessibility method studies revealed that the side chain of the 310, but not 311 residue, is both solvent accessible and directed into the agonist-binding pocket. Also, saturation mutagenesis of both Phe310 and Phe311 revealed for the former, but not for the latter, a direct relationship between side chain volume and agonist affinity, and that aromaticity is essential for wild-type agonist binding, and for both wild-type agonist potency and efficacy. Moreover, studies of Phe310 mutants combined with a previously described constitutively active alpha1B-AR mutant, A293E, indicated that although not required for spontaneous receptor isomerization from the basal state, R, to a partially activated conformation R', interaction of Phe310 with catecholamine agonists is essential for isomerization from R' to the fully activated state, R.

2-Hydroxyphenethylamine↗

PubMed1994

Genuine and apparent cross-reaction of polyclonal antibodies to proteins and peptides.

Antiserum to a native protein may cross-react with the corresponding denatured protein or with peptides. The cross-reaction is either a genuine property of the antibodies or caused by antibodies produced against some unfolded protein contaminating the native protein used for immunization. Appropriate conformation-sensitive immunoassays must be employed to distinguish a genuine from an apparent cross-reaction. In the present study, we have analyzed critically the cross-reaction of rabbit antisera against proteins and peptides. We have distinguished between genuine and apparent cross-reaction with the help of the protein A antibody-capture ELISA, a new conformation-sensitive ELISA format. Three systems were analyzed: cross-reaction of antisera to native yeast and horse cytochrome c with unfolded apo-cytochrome c; cross-reaction of antisera to a coiled-coil leucine-zipper peptide with a homologous random-coil peptide obtained by introducing two proline residues into the leucine-zipper sequence; cross-reaction of antisera to two peptides that correspond to the N-terminal and an internal sequence of ferredoxin: NADP+ reductase (FNR), with the native enzyme. The reaction of the anti-(cytochrome c) sera was clearly due to antibodies produced against unfolded protein, it was an apparent and not a genuine cross-reaction. Furthermore, the apparently cross-reactive antibodies to horse cytochrome c did not discriminate against sequence-related proteins from dog, beef, rabbit and pigeon. In contrast, antibodies to the leucine-zipper peptide did cross-react in a genuine way with the homologous random-coil peptide, that is, the cross-reactive antibodies do not seem to have been produced against the unfolded form of the leucine-zipper peptide. Of the two anti-peptide sera the one against the unstructured and highly accessible N-terminal segment reacted strongly with the native protein. The second serum against a solvent-accessible turn-like sequence of FNR showed apparent cross-reactivity: antibodies recognizing the native protein were directed against a minor conformational isoform of the free peptide and did not react with the principal form(s) of the free peptide. The generation of cross-reactive antibodies depends on the conformational stability and integrity of the immunogen and on the molecular form of its application, i.e., free, polymerized or carrier-bound. The results clarify the different nature of cross-reactivity of antisera to proteins and peptides. This knowledge is crucial if antisera are to be used as conformation-specific probes.

Amino Acid Sequence↗

PubMed1994

Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange.

Mammalian metallothionein has been postulated to play a pivotal role in cellular zinc distribution. All seven of its metal atoms are bound with high thermodynamic stability in two clusters buried deeply in the molecule. If the protein is to function in metal delivery, there must be a biological mechanism to facilitate metal release. One means to achieve this would be a labilization of the clusters by interaction of metallothionein with an appropriate cellular ligand. To search for such a mediator, we have designed a rapid radiochromatographic method that can detect changes in the zinc content of 65Zn-labeled metallothionein in response to other biomolecules. Using this methodology, we have established that rabbit liver metallothionein 2 interacts with glutathione disulfide with concomitant release of zinc. Under conditions of pseudo-first-order kinetics, the monophasic reaction depends linearly on the concentration of glutathione disulfide in the range from 5 to 30 mM with a second-order rate constant k = 4.9 x 10(-3)s-1.M-1 (pH 8.6; 25 degrees C). Apparently, zinc release does not involve direct access of glutathione disulfide to the inner coordination sphere of the metals. Rather it appears that the solvent-accessible zinc-bound thiolates in two clefts of each domain of metallothionein [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xuong, N. H., Furey, W. F., Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] participate in a thiol/disulfide interchange with glutathione disulfide. This rate-limiting initial S-thiolation, which occurs with indistinguishable rates in both clusters, then causes the clusters to collapse and release their zinc. Such a mechanism of metal release would link the control of the metal content of metallothionein to the cellular glutathione redox status and raises important questions about the physiological implications of this observation with regard to a role of glutathione in zinc metabolism and in making zinc available for other biomolecules.

Animals↗

PubMed1998

The mechanism for low-pH-induced clustering of phospholipid vesicles carrying the HA2 ectodomain of influenza hemagglutinin.

Homotrimeric hemagglutinin (HA) is one of the major spike membrane glycoproteins of the influenza virus. Initial pH-triggered conformational changes in the target membrane-interacting HA2 domain are necessary for a preliminary step in membrane fusion. Using spin-labeling electron paramagnetic resonance (EPR) spectroscopy, we examined subsequent pH-dependent changes of a membrane-bound HA2 construct (FHA2, aa 1-127). Residues 91-94, 108-115, 122, and 125 were mutated to cysteine and spin-labeled. Low solvent accessibility and side chain mobility were observed by EPR at positions 91-94, 122, and 125. Spin-labels at residues 108-115 were solvent-exposed and highly mobile, revealing the presence of a flexible loop. These results are consistent with the low-pH crystal structure of a truncated HA2 domain, particularly the unusual kink loop at residues 108-115 [Bullough et al. (1994) Nature (London) 371, 37-43]. Most interestingly, at endosomal pH, spin-labels at 108-115 become immobile and no longer solvent-exposed, and this change is reversible upon reneutralization. However, little change in the EPR line shape and accessibility of spin-labels was observed in other regions. This observation implies that the FHA2 trimers interact reversibly via this specific loop, most likely in an intermolecular fashion. Furthermore, this interaction correlates well with a reversible pH-dependent clustering of FHA2-bearing vesicles evidenced by the reversible increase in turbidity and further confirmed in detail by electron microscopy. The implications of this reversible, pH-dependent interaction between FHA2 trimers are discussed in light of recent fusion models.

Cysteine↗

PubMed1998

Structure of cubic insulin crystals in glucose solutions.

X-ray structures of cubic insulin crystals in high concentrations of glucose at different pH levels and temperatures have been refined to high resolution. We have identified one glucose-binding site near the N-terminus of the A-chain whose occupancy is pH dependent. The effects of reduced water activity on the ordered protein and solvent structures have been examined. Our analysis showed no notable conformational changes in the ordered protein structures or ordered solvent molecules near the protein surface, but the presence of glucose does have a significant effect on the overall density distribution of the bulk solvent in the solvent-accessible volume. We compared the structure of cubic insulin at room temperature and liquid-nitrogen temperature, under identical solvent conditions, using glucose as a cryoprotectant. In this case, we found that the average temperature factor of the protein is reduced and more water molecules can be identified, but there are no significant changes in the protein conformation.

Animals↗

PubMed1997

Characterization of the two myotoxin a isomers from the prairie rattlesnake (Crotalus viridis viridis) by capillary zone electrophoresis and fluorescence quenching studies.

The two myotoxin a isomers from the venom of the prairie rattlesnake Crotalus viridis viridis have different isoelectric points, as determined by capillary zone electrophoresis. The pI values are 10.50 and 10.57, respectively, and both are higher than the previously reported pI value for myotoxin a. The difference in the isoelectric points between the two isomers is attributed to altered surface charge as a result of the conformational change in myotoxin a. Both isomers exist in crude venom, discounting the possibility that they are artifacts formed during the purification process. Fluorescence quenching of myotoxin a reveals heterogeneity of the tryptophans, possibly due to different environments. The fraction of the total tryptophan fluorescence quenched by iodide is 81% and is attributed to solvent-accessible tryptophan residues at the protein surface. The 19% non-quenchable tryptophans probably represent residues that are shielded from the solvent exposure. The ratio of buried to exposed tryptophans is similar to the ratio of isomers seen by capillary zone electrophoresis and reverse-phase high-performance liquid chromatography (c. 1 : 4).

Amino Acid Sequence↗

PubMed1997

Protein solution structure calculations in solution: solvated molecular dynamics refinement of calbindin D9k.

The three-dimensional solution structures of proteins determined with NMR-derived constraints are almost always calculated in vacuo. The solution structure of (Ca2+)2-calbindin D9k has been redetermined by new restrained molecular dynamics (MD) calculations that include Ca2+ ions and explicit solvent molecules. Four parallel sets of MD refinements were run to provide accurate comparisons of structures produced in vacuo, in vacuo with Ca2+ ions, and with two different protocols in a solvent bath with Ca2+ ions. The structural ensembles were analyzed in terms of structural definition, molecular energies, packing density, solvent-accessible surface, hydrogen bonds, and the coordination of calcium ions in the two binding loops. Refinement including Ca2+ ions and explicit solvent results in significant improvements in the precision and accuracy of the structure, particularly in the binding loops. These results are consistent with results previously obtained in free MD simulations of proteins in solution and show that the rMD refined NMR-derived solution structures of proteins, especially metalloproteins, can be significantly improved by these strategies.

Calbindins↗

PubMed1983

Normal mode paths for hydrogen exchange in the peptide ferrichrome.

Possible paths for exposure to solvent and hydrogen exchange of the amide protons of ferrichrome, a cyclic hexapeptide, are examined. The paths are obtained from calculations of the vibrational normal modes of ferrichrome and correspond to low energy atomic displacements away from the local minimum in the multidimensional conformational space of the molecule. Exposure of exchangeable groups along the normal modes was determined by using the solvent accessible surface area algorithm of Lee and Richards. Three of the exchangeable protons (Gly(1,2,3,)) are largely exposed to solvent in the x-ray structure while the remaining three exchangeable protons of the ornithines are totally shielded from solvent. A very small number of normal mode displacements are found to expose the Orn(2) and Orn(3) amide groups while the Orn(1) amide proton remains shielded from solvent for all the paths studied. The effective paths for exposure of Orn(2) and Orn(3) correspond to the lowest frequency ( approximately 18 cm(-1)) motions. The paths are characterized in terms of the magnitude and energy of atomic displacements, correlated changes in dihedral angles, and the resulting changes in exposure and hydrogen bonding of exchangeable groups.

Journal Article↗

PubMed2000

A conformation change in the carboxyl terminus of Alzheimer's Abeta (1-40) accompanies the transition from dimer to fibril as revealed by fluorescence quenching analysis.

Alzheimer's disease is characterized by the presence of insoluble, fibrous deposits composed principally of amyloid beta (Abeta) peptide. A number of studies have provided information on the fibril structure and on the factors affecting fiber formation, but the details of the fibril structure are not known. We used fluorescence quenching to investigate the solvent accessibility and surface charge of the soluble Abeta(1-40) dimer and amyloid fibrils. Analogs of Abeta(1-40) containing a single tryptophan were synthesized by substituting residues at positions 4, 10, 34, and 40 with tryptophan. Quenching measurements in the dimeric state indicate that the amino-terminal analogs (AbetaF4W and AbetaY10W) are accessible to polar quenchers, and the more carboxyl-terminal analog AbetaV34W is less accessible. AbetaV40W, on the other hand, exhibits a low degree of quenching, indicating that this residue is highly shielded from the solvent in the dimeric state. Correcting for the effect of reduced translational and rotational diffusion, fibril formation was associated with a selective increase in solvent exposure of residues 34 and 40, suggesting that a conformation change may take place in the carboxyl-terminal region coincident with the dimer to fibril transition.

Alzheimer Disease↗

PubMed1999

Homogenization and crystallization of histidine ammonia-lyase by exchange of a surface cysteine residue.

Histidase (histidine ammonia-lyase, EC 4.3.1.3) from Pseudomonas putida was expressed in Escherichia coli and purified. In the absence of thiols the tetrameric enzyme gave rise to undefined aggregates and suitable crystals could not be obtained. The solvent accessibility along the chain was predicted from the amino acid sequence. Among the seven cysteines, only one was labeled as 'solvent-exposed'. The exchange of this cysteine to alanine abolished all undefined aggregations and yielded readily crystals diffracting to 1.8 A resolution.

Crystallography↗

PubMed2005

High-resolution H/D exchange studies on the HET-s218-295 prion protein.

In a search for improved resolution of hydrogen/deuterium (H/D) exchange experiments analyzed by mass spectrometry (HXMS), we evaluated two methodologies for a detailed structural study of solvent accessibility in the case of the HET-s(218-295) prion protein. For the first approach, after incubation in the deuterated solvent, aggregated HET-s(218-295) was digested with pepsin and the generated peptides were analyzed by nanospray mass spectrometry in an ion trap, with and without collision-induced dissociation (CID). We compared deuterium incorporation in peptides as determined on peptide pseudomolecular ions and on b and y fragments produced by longer peptides under CID conditions. For both b and y fragment ions, an extensive H/D scrambling phenomenon was observed, in contrast with previous studies comparing CID-MS experiments and (1)H NMR data. Thus, the spatial resolution of HXMS experiments could not be improved by means of MS/MS data generated by an ion trap mass spectrometer. In a second approach, the incorporation of deuterium was analyzed by MS for 76 peptides of the HET-s(218-289) peptide mass fingerprint, and the use of shared boundaries among peptic peptides allowed us to determine deuteration levels of small regions ranging from one to four amino acids. This methodology led to evidence of highly protected regions along the HET-s(218-295) sequence.

Amino Acid Sequence↗

PubMed1995

Alanine scanning mutagenesis of the alpha-helix 115-123 of phage T4 lysozyme: effects on structure, stability and the binding of solvent.

A series of individual alanine mutations has been constructed in the helical region 115 to 123 in phage T4 lysozyme in order to evaluate the contribution to protein stability of the different side-chains within this region. Pairwise alanine mutations and a combination mutant with seven alanine substitutions were constructed to evaluate the additive effects upon structure and stability. Only three residues within this region (Ser117, Leu118 and Leu121) have a substantial influence upon stability (change in free energy of unfolding greater than 1.0 kcal/mol). Replacement of Ser117 with alanine results in an increase in protein stability of 1.27 kcal/mol, apparently due to the release of strain present in the wild-type protein. Replacement of the buried residues Leu118 and Leu121 is destabilizing. Substitution of the remaining six residues with alanine has relatively little effect on stability. This is consistent with prior studies showing that only 20 to 30% of the residues in amphipathic helices in T4 lysozyme are critical for stability. For some of the pairwise alanine mutants the effects on stability are additive. For most of these mutants, however, there is a slight (approximately 0.15 to 0.25 kcal/mol) non-additivity such that the double mutant is more stable than the sum of the constituent single mutants. This effect is consistently observed for residues with positions i, i +4; i.e. adjacent, but in consecutive turns of the helix, suggesting a weak but significant interaction between these amino acid residues. A more pronounced non-additivity (approximately 0.5 kcal/mol) is seen in the seven-alanine combination mutant. This non-additivity is due to a modest "collapse" or "repacking" that occurs for the combination mutants (especially the multiple alanine mutant) but is not possible for the single replacements. The truncation of some side-chains permits an increase in solvent accessibility of main-chain amide and carbonyl groups. This effect is most pronounced for the seven-alanine combination mutant, where two solvent molecules, not present in wild-type, hydrogen bond to main-chain carbonyl groups in the middle region of the helix. It has been suggested that the binding of such water molecules might represent the first step in solvent-mediated unfolding of an alpha-helix. The appearance of ordered solvent, however, appears to have very little effect on stability (approximately less than 0.2 kcal/mol).

Alanine↗

PubMed2006

Hydrophobic collapse in late-stage folding (in silico) of bovine pancreatic trypsin inhibitor.

Hydrophobic collapse is commonly considered as a process of significance for protein folding. Many models have been applied for description of this event. A model introducing an external force field mimicking the hydrophobic environment and simultaneously the driving force for the folding process is presented in this paper. Bovine pancreatic trypsin inhibitor (BPTI) was taken as a test protein. An early-stage folding (in silico) model presented elsewhere was used to create the starting structure for hydrophobic collapse. The resulting structure was energy-refined using molecular dynamics simulation in an explicit solvent. The similarity versus the crystal structure of BPTI is estimated using visual analysis, residue-residue contacts, phi, psi angle distributions, RMSD, accessible solvent area, radii of gyration and hydrodynamic radii. A program allowing creation of early-stage folding structural forms to be created for any protein is available from http://bioinformatics.cm-uj.krakow.pl/earlystage. The program for late-stage folding simulation is available on request.

Animals↗

PubMed2002

Quantum mechanical/molecular mechanical free energy simulations of the glutathione S-transferase (M1-1) reaction with phenanthrene 9,10-oxide.

Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide substrates. In the present study, combined quantum mechanical/molecular mechanical (QM/MM) simulations of the conjugation of glutathione to phenanthrene 9,10-oxide, catalyzed by the M1-1 isoenzyme from rat, have been carried out to obtain insight into details of the reaction mechanism and the role of solvent present in the highly solvent accessible active site. Reaction-specific AM1 parameters for sulfur have been developed to obtain an accurate modeling of the reaction, and QM/MM solvent interactions in the model have been calibrated. Free energy profiles for the formation of two diastereomeric products were obtained from molecular dynamics simulations of the enzyme, using umbrella sampling and weighted histogram analysis techniques. The barriers (20 kcal/mol) are in good agreement with the overall experimental rate constant and with the formation of equal amounts of the two diastereomeric products, as experimentally observed. Along the reaction pathway, desolvation of the thiolate sulfur of glutathione is observed, in agreement with solvent isotope experiments, as well as increased solvation of the epoxide oxygen of phenanthrene 9,10-oxide, illustrating an important stabilizing role for active site solvent molecules. Important active site interactions have been identified and analyzed. The catalytic effect of Tyr115 through a direct hydrogen bond with the epoxide oxygen of the substrate, which was proposed on the basis of the crystal structure of the (9S,10S) product complex, is supported by the simulations. The indirect interaction through a mediating water molecule, observed in the crystal structure of the (9R,10R) product complex, cannot be confirmed to play a role in the conjugation step. A selection of mutations is modeled. The Asn8Asp mutation, representing one of the differences between the M1-1 and M2-2 isoenzymes, is identified as a possible factor contributing to the difference in the ratio of product formation by these two isoenzymes. The QM/MM reaction pathway simulations provide new and detailed insight into the reaction mechanism of this important class of detoxifying enzymes and illustrate the potential of QM/MM modeling to complement experimental data on enzyme reaction mechanisms.

Binding Sites↗

PubMed2006

Residues in substrate proteins that interact with GroEL in the capture process are buried in the native state.

We have used a bioinformatic approach to predict the natural substrate proteins for the Escherichia coli chaperonin GroEL based on two simple criteria. Natural substrate proteins should contain binding motifs similar in sequence to the mobile loop peptide of GroES that displaces the binding motif during the chaperonin cycle. Secondly, each substrate protein should contain multiple copies of the binding motif so that the chaperonin can perform "work" on the substrate protein. To validate these criteria, we have used a database of 252 proteins that have been experimentally shown to interact with the chaperonin machinery in vivo. More than 80% are identified by these criteria. The binding motifs of all 79 proteins in the database with a known three-dimensional structure are buried (<50% solvent-accessible surface area) in the native state. Our results show that the binding motifs are inaccessible in the native state but become solvent-exposed in unfolded state, thus enabling GroEL to distinguish between unfolded and native states. The structures of the binding motif in the native states of the substrate proteins include alpha-helices, beta-strands, and random coils. The diversity of secondary structures implies that there are large and varied conformational transitions in the recognition motifs after their displacement by the mobile loops of GroES.

Binding Sites↗

PubMed1998

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.

Bacterial Proteins↗

PubMed1997

Probing the cysteine 34 residue in human serum albumin using fluorescence techniques.

The microenvironment surrounding Cys-34 in human serum albumin (HSA) has been studied using acrylodan, a cysteine-specific fluorescence probe. Fluorescence emission maximum (Em(max)) of acrylodan covalently attached to Cys-34 was observed at 476 nm, which was nearly the same as for acetonitrile. The Em(max) exhibited a shift toward longer wavelength with oleate binding. The acrylodan fluorescence of HSA exhibited heterogeneous decay kinetics, which adequately fit a double-exponential decay model. When three or more oleate molecules were bound to one albumin molecule, the fractional intensity was shifted in favor of the shorter lived component. These results suggest that oleate binding induces a conformational or dynamic change which is localized in the Cys-34 region. Unfolding studies with guanidine-HCl indicate that Cys-34 appears to be located on the surface of HSA molecule and that it is protected by adjacent amino acid residues. Solvent accessibility of acrylodan with HSA in the absence and presence of oleate was determined from acrylamide quenching, and suggests that oleate binding enhances the solvent exposure of the acrylodan fluorophore. In order to determine the nature of the electrostatic potential near Cys-34, the quenching rate constants for anionic (iodide) and cationic (thallium) quenchers were determined as a function of ionic strength of solvent. The ionic strength dependence of quenching indicated that there was an electrostatic attractive force between the fluorophore and both ionic quenchers. These results are consistent with a model in which amphoteric charges which arise from charged amino acid residues were surrounding Cys-34. Interestingly, oleate binding resulted in changes in the spatial relationships between acrylodan and these charged residues. Thus, the experiments described herein provide the information concerning an oleate-induced alteration in the nature of the local environment surrounding Cys-34 and suggests that long chain fatty acid binding provides a method for regulating the radical-trapping antioxidant activity of Cys-34 in HSA in vivo.

2-Naphthylamine↗