Conformational Changes In Presence Research Articles

Biophysical Characterization of Interaction between E. coli Alanyl-tRNA Synethase with its Promoter DNA.

Aminoacyl-tRNA Synthetases (aaRSs) are well known for their role in the translation process. Lately investigators have discovered that this family of enzymes are also capable of executing a broad repertoire of functions that not only impact protein synthesis, but extend to a number of other activities. Till date, transcriptional regulation has so far only been described in E. coli Alanyl-tRNA synthetase and it was demonstrated that alaRS binds specifically to the palindromic DNA sequence flanking the gene's transcriptional start site and thereby regulating its own transcription. In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques. To understand the role of full length protein and oligomerization of alaRS in promoter DNA binding, two mutants were constructed, namely, N700 (a monomer, containing the N-terminal aminoacylation domain but without the C-terminal part) and G674D (previously demonstrated to form full-length monomer). Protein-DNA binding study using fluorescence spectroscopy, analytical ultracentrifugation, Isothermal Titration Calorimetry was conducted. Sedimentation equilibrium studies clearly demonstrated that monomeric variants were unable to bind promoter DNA. Isothermal Calorimetry (ITC) experiment was employed for further characterization of wild type alaRS-DNA interaction. It was observed that full length E. coli Alanyl-tRNA synthetase binds specifically with its promoter DNA and forms a dimer of dimers. On the other hand the two mutant variants were unable to bind with the DNA. In this study it was concluded that full length E. coli Alanyl-tRNA synthetase undergoes a conformational change in presence of its promoter DNA leading to formation of higher order structures. However, the exact mechanism behind this binding is currently unknown and beyond the scope of this study.

Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705-SH2 through C-terminal tail modulation.

Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705-SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT-CTT intermolecular and the CTT-SH2 intramolecular interactions that support pY705-SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705-SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705-SH2 interaction. Thus, pS727 triggers pY705-SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation-inactivation for proper function of STAT3.

Theory of Diffusion-Influenced Reaction Networks.

A formalism is developed to describe how diffusion alters the kinetics of coupled reversible association-dissociation reactions in the presence of conformational changes that can modify the reactivity. The major difficulty in constructing a general theory is that, even to the lowest order, diffusion can change the structure of the rate equations of chemical kinetics by introducing new reaction channels (i.e., modifies the kinetic scheme). Therefore, the right formalism must be found that allows the influence of diffusion to be described in a concise and elegant way for networks of arbitrary complexity. Our key result is a set of non-Markovian rate equations involving stoichiometric matrices and net reaction rates (fluxes), in which these rates are coupled by a time-dependent pair association flux matrix, whose elements have a simple physical interpretation. Specifically, each element is the probability density that an isolated pair of reactants irreversibly associates at time t via one reaction channel on the condition that it started out with the dissociation products of another (or the same) channel. In the Markovian limit, the coupling of the chemical rates is described by committors (or splitting/capture probabilities). The committor is the probability that an isolated pair of reactants formed by dissociation at one site will irreversibly associate at another site rather than diffuse apart. We illustrate the use of our formalism by considering three reversible reaction schemes: (1) binding to a single site, (2) binding to two inequivalent sites, and (3) binding to a site whose reactivity fluctuates. In the first example, we recover the results published earlier, while in the second one we show that a new reaction channel appears, which directly connects the two bound states. The third example is particularly interesting because all species become coupled and an exchange-type bimolecular reaction appears. In the Markovian limit, some of the diffusion-modified rate constants that describe new transitions become negative, indicating that memory effects cannot be ignored.

Versatile multicharacterization platform involving tailored superhydrophobic SU-8 micropillars for the investigation of breast cancer estrogen receptor isoforms

Here, the authors report the fabrication of lotus-leaf-like tailored SU8 micropillars and their application in the context of a multitechnique characterization protocol for the investigation of the structural properties of the two estrogen receptors (ERα66/ERα46). ER (α) expression is undoubtedly the most important biomarker in breast cancer, as it provides the index for sensitivity to endocrine treatment. Beside the well-characterized ERα66 isoform, a shorter one (ERα46) is also expressed in ERα positive breast cancers and breast cancer cell lines. The superhydrophobic supports were developed by using a two-step approach including an optical lithography process and a plasma reactive ion roughening one. Upon drying on the micropillars, the biological samples resulted in stretched fibers of different diameters which were then characterized by synchrotron x-ray diffraction (XRD), Raman and Fourier-transform infrared spectroscopy. The evidence of both different spectroscopic vibrational responses and XRD signatures in the two estrogen receptors suggests the presence of conformational changes between the two biomarkers. The SU8 micropillar platform therefore represents a valid tool to enhance the discrimination sensitivity of structural features of this class of biomarkers by exploiting a multitechnique in situ characterization approach.

Conformation-independent structural comparison of macromolecules with ProSMART

Structural comparison often aids insight into the function and mechanics of biological macromolecules. To make such analyses more accessible, we present the Procrustes Structural Matching Alignment and Restraints Tool (ProSMART), which is designed to allow fast but detailed comparative analysis of macromolecular structures despite potential dissimilarities in global arrangement, such as domain motion and distortion. Whilst obtaining a residue alignment between structures is a prerequisite for comparative analysis, conventional alignment methods may fail in cases where conformational differences are dramatic. However, ProSMART achieves a conformation-independent structural alignment by focusing purely on local dissimilarities, rather than enforcing chain/domain rigidity. This allows the sensible comparison of protein (or DNA/RNA) structures in the presence of conformational change. ProSMART allows analysis of the structural conservation of local backbone and side chains in a wide variety of scenarios - the method is sensitive enough to allow identification of subtle dissimilarities between structures sharing high sequence homology, whilst being versatile enough to allow identification of local similarities between more distantly-related structures. In addition, ProSMART can be used for the identification of conserved rigid substructures, which may or may not represent functional domains. ProSMART is also used for the generation of external restraints for use in crystallographic refinement. Results from ProSMART can be visualised in either CCP4mg or PyMOL. All residue-based scores are illustrated using intuitive colour gradients, allowing easy visual assessment of local backbone and side chain conservation. Complementary structural comparison tools such as ProSMART can help break the complexity of the constantly growing pool of available structural data into a more readily accessible form, and consequently may aid biological insight into macromolecular structures.

Introducing Human Serum Albumin as a Potent Flexible Protein in Presence of Drugs and Ligands

Human serum albumin (HAS) plays important roles in the body including transport of different ions, fatty acids, drugs, hormones and metabolites. This protein has a key role in regulation of blood osmotic pressure. In correspond to the wide variations of functional aspects of HSA, its structure is characterized by various conformations. In this study, HSA behavior during denaturation and conformational changes in presence of flouxetine and cortisol (as ligands) at 37oC and 42oC temperatures is studied by using pH metery denaturation, UV spectroscopy, florescence spectroscopy, and circular dichroism (CD) techniques. Differential scanning calorimetery profiles of HSA in the absence and presence of ligands are also provided and have been analyzed. Complicated statistical analysis shows the existence of several fine structures features for HSA. This ability of HSA for structural switching corresponds to potent potential of albumin for doing various functions. The finding can be attributed to the other similar proteins by additional investigations.Keywords: Human Serum Albumin, Conformation, Fine Structural Features, Conformer and Denaturation

Functional role of domains in the Snf2 subunit of the SWI/SNF complex outside the catalytic domain

SWI/SNF is the classical ATP dependent chromatin remodeler that restructures nucleosomes by virtue of its DNA and nucleosome stimulated ATPase activity. In this context, much attention has been devoted to the biochemical and genetic analyses of the ATPase‐helicase domain of the catalytic subunit Snf2. The catalytic contribution of the regions outside this domain has been explored to a limited extent, mostly focusing on the C terminal bromodomain. In this study, we use bioinformatic tools to identify putative domains in Snf2 (N, QLQ, spacer and AT), selectively delete them and characterize the domain deleted complexes both in vitro and in vivo. Our results indicate that complex integrity is preserved in the absence of these domains. Deletion of N terminus and the double AT hook show no difference in activity in vitro although these deletions have a snf2 null global gene expression pattern. In contrast, "QLQ" and "spacer" deletions are catalytically defective both in vitro and in vivo. In vitro, they show poor ATP hydrolysis and remodeling capacity. In vivo, they closely mimic snf2 null phenotype. Site directed DNA and histone crosslinking studies identify subtle conformational changes in presence of these deletions that may perturb proper nucleosomal contacts critical for remodeling. In summary, regions outside the ATPase‐helicase domain of Snf2 contribute positively to the catalytic function of the holocomplex.

Polymer-induced heteronucleation of tolfenamic acid: structural investigation of a pentamorph.

The nonsteroidal anti-inflammatory drug (NSAID) tolfenamic acid (TA), previously thought to be dimorphic, is demonstrated to have at least five polymorphs. The new forms were uncovered through the emerging screening technique of polymer-induced heteronucleation (PIHn). The presence of conformational changes among forms, whole molecule disorder, space group diversity, and varying number of molecules in the asymmetric unit occurring within a very narrow free energy window (approximately 0.3 kcal/mol) make the solid-state chemistry of this molecule uniquely complex among pharmaceuticals. These aspects make it a particularly suitable benchmark compound for crystal structure prediction methods.

Alignment of protein structures in the presence of domain motions

BackgroundStructural alignment is an important step in protein comparison. Well-established methods exist for solving this problem under the assumption that the structures under comparison are considered as rigid bodies. However, proteins are flexible entities often undergoing movements that alter the positions of domains or subdomains with respect to each other. Such movements can impede the identification of structural equivalences when rigid aligners are used.ResultsWe introduce a new method called RAPIDO (Rapid Alignment of Proteins in terms of Domains) for the three-dimensional alignment of protein structures in the presence of conformational changes. The flexible aligner is coupled to a genetic algorithm for the identification of structurally conserved regions. RAPIDO is capable of aligning protein structures in the presence of large conformational changes. Structurally conserved regions are reliably detected even if they are discontinuous in sequence but continuous in space and can be used for superpositions revealing subtle differences.ConclusionRAPIDO is more sensitive than other flexible aligners when applied to cases of closely homologues proteins undergoing large conformational changes. When applied to a set of kinase structures it is able to detect similarities that are missed by other alignment algorithms. The algorithm is sufficiently fast to be applied to the comparison of large sets of protein structures.

RAPIDO: a web server for the alignment of protein structures in the presence of conformational changes

Rapid alignment of proteins in terms of domains (RAPIDO) is a web server for the 3D alignment of crystal structures of different protein molecules in the presence of conformational change. The structural alignment algorithm identifies groups of equivalent atoms whose interatomic distances are constant (within a defined tolerance) in the two structures being compared and considers these groups of atoms as rigid bodies. In addition to the functionalities provided by existing tools, RAPIDO can identify structurally equivalent regions also when these consist of fragments that are distant in terms of sequence and separated by other movable domains. Furthermore, RAPIDO takes the variation in the reliability of atomic coordinates into account in the comparison of distances between equivalent atoms by employing weighting-functions based on the refined B-values. The regions identified as equivalent by RAPIDO furnish reliable sets of residues for the superposition of the two structures for subsequent detailed analysis. The RAPIDO server, with related documentation, is available at http://webapps.embl-hamburg.de/rapido.

Diversity of Penicillin-binding Proteins

Antibiotic-resistant Staphylococcus aureus is a major concern to public health. Methicillin-resistant S. aureus strains are completely resistant to all beta-lactams antibiotics. One of the main factors involved in methicillin resistance in S. aureus is the penicillin-binding protein, PBP2a. This protein is insensitive to inactivation by beta-lactam antibiotics such as methicillin. Although other proteins are implicated in high and homogeneous levels of methicillin resistance, the functions of these other proteins remain elusive. Herein, we report for the first time on the putative function of one of these proteins, FmtA. This protein specifically interacts with beta-lactam antibiotics forming covalently bound complexes. The serine residue present in the sequence motif Ser-X-X-Lys (which is conserved among penicillin-binding proteins and beta-lactamases) is the active-site nucleophile during the formation of acyl-enzyme species. FmtA has a low binding affinity for beta-lactams, and it experiences a slow acylation rate, suggesting that this protein is intrinsically resistant to beta-lactam inactivation. We found that FmtA undergoes conformational changes in presence of beta-lactams that may be essential to the beta-lactam resistance mechanism. FmtA binds to peptidoglycan in vitro. Our findings suggest that FmtA is a penicillin-binding protein, and as such, it may compensate for suppressed peptidoglycan biosynthesis under beta-lactam induced cell wall stress conditions.

Substrate-Induced Conformational Changes of Melibiose Permease from Escherichia coli Studied by Infrared Difference Spectroscopy

Fourier transform infrared difference spectroscopy has been used to obtain information about substrate-induced structural changes of the melibiose permease (MelB) from Escherichia coli reconstituted into liposomes. Binding of the cosubstrate Na(+) gives rise to several peaks in the amide I and II regions of the difference spectrum Na(+).MelB minus H(+).MelB, that denote the presence of conformational changes in all types of secondary structures (alpha-helices, beta-sheets, loops). In addition, peaks around 1400 and at 1740-1720 cm(-1) are indicative of changes in protonation/deprotonation or in environment of carboxylic groups. Binding of the cosubstrate Li(+) produces a difference spectrum that is also indicative of conformational changes, but that is at variance as compared to that induced by Na(+) binding. To analyze the following transport steps, the melibiose permease with either H(+), Na(+), or Li(+) bound was incubated with melibiose. The difference spectra obtained by subtracting the spectrum cation.MelB from the respective complex cation.melibiose.MelB were roughly similar among them, but different from those induced by cation binding, and more intense. Therefore, major conformational changes that are induced during melibiose binding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm(-)(1), are similar for the three cotransporting cations. Changes in the protonation state and/or in the environment of given carboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.

The Density and Refractive Index of Adsorbing Protein Layers

The structure of the adsorbing layers of native and denatured proteins (fibrinogen, γ-immunoglobulin, albumin, and lysozyme) was studied on hydrophilic TiO 2 and hydrophobic Teflon-AF surfaces using the quartz crystal microbalance with dissipation and optical waveguide lightmode spectroscopy techniques. The density and the refractive index of the adsorbing protein layers could be determined from the complementary information provided by the two in situ instruments. The observed density and refractive index changes during the protein-adsorption process indicated the presence of conformational changes (e.g., partial unfolding) in general, especially upon contact with the hydrophobic surface. The structure of the formed layers was found to depend on the size of the proteins and on the experimental conditions. On the TiO 2 surface smaller proteins formed a denser layer than larger ones and the layer of unfolded proteins was less dense than that adsorbed from the native conformation. The hydrophobic surface induced denaturation and resulted in the formation of thin compact protein films of albumin and lysozyme. A linear correlation was found between the quartz crystal microbalance measured dissipation factor and the total water content of the layer, suggesting the existence of a dissipative process that is related to the solvent molecules present inside the adsorbed protein layer. Our measurements indicated that water and solvent molecules not only influence the 3D structure of proteins in solution but also play a crucial role in their adsorption onto surfaces.

Structural Insights into Molecular Function of the Metastasis-associated Phosphatase PRL-3

Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys(49) that forms an intramolecular disulfide bond with the catalytic Cys(104) even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence.

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

NMR studies of the metal-loading kinetics and acid-base chemistry of DOTA and butylamide-DOTA.

The conjugation of a chelating agent to a protein via a covalent linkage has been previously reported to change the metal-binding characteristics of the chelator. A fundamental understanding of these binding changes would enable design of a new generation of metal-chelating agents for biological applications. To assess the effect of conjugation on the commonly used chelating agent 1 4,7, 10-tetraaazacyclododecane-N,N',N'',N'''-tetraaacetic acid (DOTA), we synthesized a model protein conjugate, 1,4, 7-tris(carboxymethyl)-10-(butylaminocarboxymethyl)-1,4,7, 10-tetraaazacyclododecane (BD) and explored the metal-binding characteristics via NMR. The extent of ionization of the carboxylic acid groups and the two protonated macrocycle nitrogens of DOTA and BD were determined as a function of pH by chemical shift changes in proximal carbon-bonded protons. In addition to the expected sensitivity of the chemical shifts to titration of proximate acidic groups, BD resonances from carbon-bonded protons 10-17 bonds distant from the deprotonation site also shifted significantly, indicating the presence of conformational changes. Furthermore, increased shielding of the amide and alkyl proton signals upon deprotonation of the carboxylic acid groups indicates the presence of pH-dependent hydrogen-bonded BD isoforms. On the basis of these NMR data, we propose new structures for the doubly protonated forms of DOTA and BD. To measure metal loading, the yttrium-loading rates (type I to type II) of DOTA and BD were determined by following the intensity of type I and type II proton signals as a function of time. The yttrium-loading rates of BD are approximately one-half those of DOTA at pHs between 4.6 and 6.5 and 37 degrees C. The loading rates measured as a function of pH indicate that while both the doubly protonated and singly protonated forms of DOTA are reactive to metal loading, only the singly protonated form of BD is reactive.

Ultrasonic absorption in solutions of homo‐ and copolymers of vinyl chloride and vinyl acetate

AbstractThe ultrasonic attenuation in solutions of poly(vinyl chloride), poly(vinyl acetate) and different vinyl chloride/vinyl acetate copolymers dissolved in cyclohexanone was investigated. The results indicate that the relaxation spectra can be resolved by curves with two relaxation times. These processes can be explained by the presence of conformational changes of different degree of cooperativity. Comparison of the thermodynamic parameters with conformational energy calculations supports the assumption that the elementary process is a localized single‐bond process. The ultrasonic spectra of the homopolymers solutions are compared with those for statistical and graft copolymers.

Hydrogen—tritium exchange of rhodopsin: Effect of solvent on the incorporation of slowly exchanging tritium atoms

Rhodopsin, the visual pigment of the vertebrate retinal rod outer segments undergoes isomerisation of its chromophoretic group, retinal, on absorption of a photon. It has been widely suggested that after photoisomerisation of the retinal, rhodopsin is responsible for the release of a transmitter into the interdiscal space of the rod outer segment [ 11. However this transmitter has not yet been identified; indeed it is not yet clear to what extent rhodopsin’s polypeptide chain undergoes conformational changes upon photoisomerisation of the retinal. The hydrogen-tritium exchange technique has often been used to demonstrate the presence of conformational changes in proteins [2,3]. They are visualised as changes in the exchange kinetics of the proteins labile hydrogens. However in order that a conformational change can be detected the labile protons of the polypeptide chain, associated with the conformational change considered, must have become tritiated during the exchange-in of incubation time. Parameters such as the pH, temperature, ionic strength, and the composition of the medium will change the freely accessible proton exchange rate [2] and/or the conformation of the protein and hence modify the observed exchange kinetics. Thus in order that the conformational changes of rhodopsin upon illumination can be studied it is necessary to ensure that the associated labile hydrogens have become tritiated during the incubation time. The effect of several incubation

Identification of chloride-binding sites in hemoglobin by nuclear-magnetic-resonance quadrupole-relaxation studies of hemoglobin digests.

35Cl minus-nuclear magnetic resonance (NMR) studies indicate that various digests of human hemoglobin with carboxypeptidase A and B, or a combination of the two, may be used for the identification of chloride binding sites. All the digestion products contain, like hemoglobin itself, at least two classes of binding sites, one of high, the others of low affinity. The pH dependence of the excess linewidth of the 35Cl minus NMR signal indicates that in the simple digests with either carboxypeptidase A or B, chloride is bound with high affinity at or near His-beta146-Asp-beta94 and at or near Val-alpha1-Arg-alpha141. The high-affinity sites show, in the case of the simple digests, a strong oxygen linkage which is lost in the forms digested with both carboxypeptidase A and B; this linkage may thus be correlated to the presence of conformational changes. Organic phosphates, like inositol hexaphosphate, show competition for some of the high-affinity chloride binding sites in hemoglobin and in the simple digests. This competition is likewise lost in the doubly digested hemoglobins.

Kinetics of opiate receptor inactivation by sulfhydryl reagents: evidence for conformational change in presence of sodium ions.

The role of SH groups in opiate-receptor interactions has been further examined. In activation by N-ethylmaleimide of sterospecific opiate binding by rat brain membrane fractions follows pseudo-first order kinetics and exhibits strong temperature dependence. The kinetics indicate that alkylation of a single SH group suffices to block opiate binding. Considerable protection from SH group inactivation is observed when treatment with N-ethylmaleimide is carried out in the presence of an opiate or an antagonist, suggesting close proximity of the SH group to the opiate binding site. The rate of inactivation of receptor binding by N-ethylmaleimide is markedly slower in buffers containing 100 mM NaCl (t1/2 equals 30 plus or minus 1.4 min) than in sodium-free buffers (t1/2 equals 10 plus or minus 1.0 min). Since the rate of alkylation of model SH compounds is unaffected by sodium ions, this protection seems best explained by a conformational change in the receptors that renders the SH groups less accessible to alkylation. The rate of inactivation is not affected by K+, Rb+, or Cs+ and only slightly by Li+. This cation specificity as well as the concentration-response to Na+ are remarkably similar to those previously shown to lead to increased antagonist and decreased agonist binding. We suggest that the same conformational change is involved in the two phenomena.