Native Membrane Environment Research Articles

The Use of the Condensed Single Protein Production System for Isotope-Labeled Outer Membrane Proteins, OmpA and OmpX in E. coli

Gram-negative bacteria consist of two independent membranes, the inner cytoplasmic membrane and the outer membrane. The outer membrane contains a number of β-barrel proteins such as OmpF, OmpC, OmpA, and OmpX. In this article, we explored to use the condensed Single Protein Production (cSPP) system for isotope labelling of OmpA and OmpX for NMR structural study, both of which are known to consist of eight β-strands forming a barrel in the outer membrane. Using a deletion strain lacking all major outer membrane proteins, both OmpA and OmpX were expressed well in a 20-fold cSPP system. We demonstrated that outer membrane fractions prepared from the cSPP system in M9 medium containing ¹⁵N-NH₄Cl can be directly used for NMR structural study of the outer mebrane proteins without any further purification to get excellent [¹H-¹⁵N]-TROSY spectra. This method would be quite valuable for the study of pure proteins in their native membrane environment without the need of purification and reconstitution.

Calcium-dependent Conformational Changes in Inositol Trisphosphate Receptors

We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca(2+)-induced conformational changes of IP(3)Rs in their native membrane environment. We found that Ca(2+) decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca(2+) induces a conformational change in the IP(3)R that allows trypsin to cleave the C-terminal epitope. Half-maximal effects of Ca(2+) were observed at approximately 0.5 microm and was sensitive to inhibition by IP(3). Ca(2+) also stimulated the reaction of MPEG-5 with an endogenous thiol in the 95-kDa fragment. This effect was eliminated when six closely spaced cysteine residues proximal to the transmembrane domains were mutated (C2000S, C2008S, C2010S, C2043S, C2047S, and C2053S) or when the N-terminal suppressor domain (amino acids 1-225) was deleted. A cysteine substitution mutant introduced at the C-terminal residue (A2749C) was freely accessible to MPEG-5 or MPEG-20 in the absence of Ca(2+). However, cysteine substitution mutants in the interior of the tail were poorly reactive with MPEG-5, although reactivity was enhanced by Ca(2+). We conclude the following: a) that large conformational changes induced by Ca(2+) can be detected in IP(3)Rs in situ; b) these changes may be driven by Ca(2+) binding to the N-terminal suppressor domain and expose a group of closely spaced endogenous thiols in the channel domain; and c) that the C-terminal cytosol-exposed tail of the IP(3)R may be relatively inaccessible to regulatory proteins unless Ca(2+) is present.

The role of individual amino acids in the dimerization of CR4 and ACR4 transmembrane domains

CRINKLY4 is a growth factor-like plant receptor kinase designated as CR4 in Zea mays and ACR4 in Arabidopsis. Using the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment, we have previously demonstrated that the dimerization potential of the ACR4 transmembrane (TM) domain is significantly weaker than that of CR4 TM domain, even though 13 of the 24 residues are identical. Neither of the TM domains contain the GxxxG motif that has been shown to be important for the dimerization of the TM segments of several receptors. To further investigate the relationship between protein sequence and dimerization potential, we (a) mutated each of the 11 differing residues in the CR4 TM domain to the corresponding residue of ACR4 (b) made reciprocal mutations in ACR4 and (c) made hybrids consisting of half CR4 and half ACR4 TM domains. Our results suggest that most mutations in ACR4 or CR4 TM domains have low to moderate effects on the dimerization potential and that residues in the N-terminal half of the CR4 TM domain are important for dimerization.

Designer Amphiphilic Short Peptides Enhance Thermal Stability of Isolated Photosystem-I

Stability of membrane protein is crucial during protein purification and crystallization as well as in the fabrication of protein-based devices. Several recent studies have examined how various surfactants can stabilize membrane proteins out of their native membrane environment. However, there is still no single surfactant that can be universally employed for all membrane proteins. Because of the lack of knowledge on the interaction between surfactants and membrane proteins, the choice of a surfactant for a specific membrane protein remains purely empirical. Here we report that a group of short amphiphilic peptides improve the thermal stability of the multi-domain protein complex photosystem-I (PS-I) in aqueous solution and that the peptide surfactants have obvious advantages over other commonly used alkyl chain based surfactants. Of all the short peptides studied, Ac-I5K2-CONH2 (I5K2) showed the best stabilizing effect by enhancing the melting temperature of PS-I from 48.0°C to 53.0°C at concentration of 0.65 mM and extending the half life of isolated PS-I significantly. AFM experiments showed that PS-I/I5K2/Triton X-100 formed large and stable vesicles and thus provide interfacial environment mimicking that of native membranes, which may partly explain why I5K2 enhanced the thermal stability of PS-I. Hydrophobic and hydrophilic group length of IxKy had an important influence on the stabilization of PS-I. Our results showed that longer hydrophobic group was more effective in stabilizing PS-I. These simple short peptides therefore exhibit significant potential for applications in membrane protein studies.

A reconstitution protocol for the in vitro folded human G protein-coupled Y2 receptor into lipid environment

Although highly resolved crystal structures of G protein-coupled receptors have become available within the last decade, the need for studying these molecules in their natural membrane environment, where the molecules are rather dynamic, has been widely appreciated. Solid-state NMR spectroscopy is an excellent method to study structure and dynamics of membrane proteins in their native lipid environment. We developed a reconstitution protocol for the uniformly 15N labeled Y2 receptor into a bicelle-like lipid structure with high yields suitable for NMR studies. Milligram quantities of target protein were expressed in Escherichia coli using an optimized fermentation process in defined medium yielding in over 10mg/L medium of purified Y2 receptor solubilized in SDS micelles. The structural integrity of the receptor molecules was strongly increased through refolding and subsequent reconstitution into phospholipid membranes. Specific ligand binding to the integrated receptor was determined using radioligand affinity assay. Further, by NMR measurement a dispersion of the 15N signals comparable to native rhodopsin was shown. The efficiency of the reconstitution could also be inferred from the fact that reasonable 13C NMR spectra at natural abundance could be acquired.

LCP-Tm: An Assay to Measure and Understand Stability of Membrane Proteins in a Membrane Environment

Structural and functional studies of membrane proteins are limited by their poor stability outside the native membrane environment. The development of novel methods to efficiently stabilize membrane proteins immediately after purification is important for biophysical studies, and is likely to be critical for studying the more challenging human targets. Lipidic cubic phase (LCP) provides a suitable stabilizing matrix for studying membrane proteins by spectroscopic and other biophysical techniques, including obtaining highly ordered membrane protein crystals for structural studies. We have developed a robust and accurate assay, LCP-Tm, for measuring the thermal stability of membrane proteins embedded in an LCP matrix. In its two implementations, protein denaturation is followed either by a change in the intrinsic protein fluorescence on ligand release, or by an increase in the fluorescence of a thiol-binding reporter dye that measures exposure of cysteines buried in the native structure. Application of the LCP-Tm assay to an engineered human β2-adrenergic receptor and bacteriorhodopsin revealed a number of factors that increased protein stability in LCP. This assay has the potential to guide protein engineering efforts and identify stabilizing conditions that may improve the chances of obtaining high-resolution structures of intrinsically unstable membrane proteins.

Complex Formation and Light Activation in Membrane-Embedded Sensory Rhodopsin II as Seen by Solid-State NMR Spectroscopy

Microbial rhodopsins execute diverse biological functions in the cellular membrane. A mechanistic understanding of their functional profile is, however, still limited. We used solid-state NMR (ssNMR) spectroscopy to study structure and dynamics of a 2 x 400 amino acid sensory rhodopsin/transducer (SRII/HtrII) complex from Natronomonas pharaonis in a natural membrane environment. We found a receptor-transducer binding interface in the ground state that significantly extends beyond the available X-ray structure. This binding domain involves the EF loop of the receptor and stabilizes the functionally relevant, directly adjacent HAMP domain of the transducer. Using 2D ssNMR difference spectroscopy, we identified protein residues that may act as a functional module around the retinal binding site during the early events of protein activation. These latter protein segments, the inherent plasticity of the HAMP domain, and the observation of an extended SRII/HtrII membrane-embedded interface may be crucial components for optimal signal relay efficiency across the cell membrane.

Hydroxide Ion Channel Controls Uncoupling and Thermogenesis of Brown Fat Mitochondria

Uncoupling proteins (UCP1- UCP5) are six-transmembrane-domain transport proteins of the inner mitochondrial membrane (IMM). They increase electrical conductance of the IMM, thus dissipating the electrochemical proton gradient across this membrane and uncoupling mitochondrial respiration and ATP synthesis. By controlling mitochondrial membrane potential, UCPs can affect many aspects of mitochondrial function and have been implicated in regulation of body's energy efficiency, reducing fat depositions, thermogenesis, diabetes, and protecting the cell against oxidative damage and ageing. The founding member of the family, UCP1, is specifically expressed in brown adipose tissue (BAT) and is responsible for adaptive thermogenesis mediated by this tissue. Due to its unusually high level of expression, upon activation UCP1 completely uncouples BAT mitochondria and converts the energy of the substrate oxidation into heat. Since UCP1 can dissipate large amounts of energy, it has attracted attention as a potential target to treat obesity. In spite of their physiological and therapeutic significance, the mechanism of operation of uncoupling proteins including their ionic selectivity has long remained unknown due to the lack of direct methods to study their activity in their native membrane environment. Here, by applying the patch-clamp technique to the whole inner membrane of BAT mitochondria and for the first time directly measuring transmembrane currents produced by UCP1, we show that UCP1 is a ligand-gated hydroxide (OH-) ion channel activated by fatty acids. UCP1 is the only hydroxide ion channel reported to date. Thus, BAT thermogenesis involves the outward transport of protons by the electron transport chain along with the outward transport of OH- by UCP1, thereby amounting to cycling of water across the IMM and not to futile cycling of protons as was largely considered before.

The Involvement of Protein-Protein Interactions in the Mechanism of the Na+,K+-ATPase

The Na+,K+-ATPase (or sodium pump) was the first ion pump to be discovered (Skou, 1957) and it is one of the most fundametally important enzymes of animal physiology. The electrochemical potential for Na+, which the enzyme maintains, is used as the driving force for numerous secondary transport systems, e.g. voltage-sensitive Na+ channels in nerve. ATP provides the energy source to drive ion pumping. However, it also plays a crucial allosteric role, accelerating significantly the enzyme's rate determining E2-E1 transition and the associated release of K+ ions to the cytoplasm. Based on the results of stopped-flow kinetic experiments and recently published crystal structural data for the related enzyme, the sarcoplasmic reticulum Ca2+-ATPase, it is suggested that the allosteric role of ATP in the mechanism of the Na+,K+-ATPase can be explained by an ATP-induced closing of the cytoplasmic domains of the enzyme which relieves steric hindrance arising from interactions between neighbouring pump molecules within the native membrane environment and hence an acceleration of the E2-E1 conformational change (Clarke, 2009). In the presence of millimolar concentrations of ATP, therefore, it is proposed that the enzyme functions as a monomer (alpha-beta protomer), whereas at low ATP concentrations it functions as a dimer ((alpha-beta)2 diprotomer) or higher aggregate. The physiological advantage of protein-protein interactions is still unclear, but a possibility is that they may lead to an enhancement of the enzyme's ATP affinity and allow it to continue functioning even under hypoxic conditions.Skou JC. (1957) Biochimica et Biophysica Acta, 23: 394-401.Clarke RJ (2009) European Biophysics Journal, in press.

Electron Microscopy of VDAC Membrane Crystals Redux. Pore Shape, Size, and Location(s) of the N-Terminal Domain

Atomic structures of mammalian VDAC, the mitochondrial outer membrane channel protein, have recently been determined by three groups using NMR and x-ray crystallography. The three structures are similar, showing a 19-strand β-barrel into which the N-terminal domain inserts. That this common protein fold was determined independently in three different labs strongly suggests that it is an easily accessible low-energy state of the protein. Nonetheless, concerns have been raised whether the atomic structures obtained using bacterially expressed proteins refolded in detergent micelles or lipid bicelles correspond to physiologically relevant states of this integral membrane protein. The definitive answer to this question will require detailed information about the topology of VDAC in its native mitochondrial outer membrane environment. Some insights can be provided by past electron microscopic (EM) studies of ordered arrays of fungal VDAC in isolated mitochondrial outer membranes. Projected density maps of the VDAC protein in frozen-hydrated membranes are consistent with a circular β-barrel having a diameter of 3.6+/-0.2 nm at the alpha-carbon backbone, in good agreement with the atomic models. The low resolution of the EM density maps may have precluded detection of the N-terminal domain within the pore lumen. However, antibodies against the N-terminal domain bound well both to isolated mitochondria and outer membranes, and the corresponding Fab fragments mapped to membrane regions adjacent to the pore. These data indicate that the N-terminus of VDAC in the mitochondrial outer membrane occurs outside the β-barrel at least some of the time. That the amphipathic N-terminal domain might move in and out of the lumen is suggested by changes in the pore detected by multivariate statistical analysis and difference imaging of VDAC arrays embedded in gold-glucose. (Supported by NSF grants and NIH/NCRR grant RR01219.)

Protein Folding at the Membrane Interface: The Structure of Nogo-66

Compelling evidence indicates that repair of damage to the central nervous system (CNS) is inhibited by the presence of protein factors within myelin that prevent axonal regrowth. Myelin growth inhibits and their common receptor have been identified as targets in the treatment of damage to the CNS.We have recently determined the NMR structure of one of the myelin growth inhibitors, the neurite outgrowth inhibitor (Nogo). We studied the structure of this protein alone and in the presence of dodecylphosphocholine micelles to mimic the natural cell membrane environment. Using several paramagnetic probes, we have defined portions of the growth inhibitor that are accessible to solvent (and consequently the Nogo receptor). Mutagenesis probed through phage-display confirms that the positions predicted to be extra-cellular are sensitive to receptor binding. Using computational docking methods and the mutagenesis results, we calculated the optimal protein-protein interface between our structure of Nogo and the Nogo receptor. The structure of Nogo and the predicted Nogo/receptor inhibitory complex structure will be presented.Funding was provided by: NIH 1R01GM078528-01 andCal Roman Reed Res Fund RR03-072, +114, +155

Structure and hydration of membranes embedded with voltage-sensing domains

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly-charged S1–S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated potassium channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1–S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations, cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings reveal that voltage sensors have evolved to interact with the lipid membrane while keeping the energetic and structural perturbations to a minimum, and that water penetrates into the membrane to hydrate charged residues and shape the transmembrane electric field.

Influence of K +-dependent membrane lipid composition on the expression of the kdpFABC operon in Escherichia coli

The membrane-bound sensor kinase KdpD and the cytoplasmic response regulator KdpE regulate the expression of the kdpFABC operon coding for the high affinity potassium uptake system KdpFABC in Escherichia coli. The signal transduction cascade of this two component system is activated under K +-limiting conditions in the medium, but is less sensitive to high osmolality. In order to test whether K + limitation affects membrane phospholipid composition and whether this change affects kdpFABC expression, we analysed the phospholipid composition of E. coli under these conditions. Our measurements revealed that there is an increase in the cardiolipin (CL) content during the exponential growth phase at the expense of the zwitterionic phospholipid phosphatidylethanolamine. The higher anionic phospholipid content occurs along with an increase of transcriptional activity of the cls gene coding for CL synthase. Furthermore, in vivo studies with E. coli derivatives carrying mutations in genes coding for enzymes involved in phospholipid biosynthesis revealed that the increase in the anionic lipid composition enhances the expression rate of the kdpFABC operon. Finally, we show that kinase activity of KdpD is stimulated in its native membrane environment by fusion with liposomes of anionic, but reduced with liposomes of zwitterionic phospholipids.

NMR and EPR studies of membrane transporters

In order to fulfill their function, membrane transport proteins have to cycle through a number of conformational and/or energetic states. Thus, understanding the role of conformational dynamics seems to be the key for elucidation of the functional mechanism of these proteins. However, membrane proteins in general are often difficult to express heterologously and in sufficient amounts for structural studies. It is especially challenging to trap a stable energy minimum, e.g., for crystallographic analysis. Furthermore, crystallization is often only possible by subjecting the protein to conditions that do not resemble its native environment and crystals can only be snapshots of selected conformational states. Nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy are complementary methods that offer unique possibilities for studying membrane proteins in their natural membrane environment and for investigating functional conformational changes, lipid interactions, substrate-lipid and substrate-protein interactions, oligomerization states and overall dynamics of membrane transporters. Here, we review recent progress in the field including studies from primary and secondary active transporters.

Assigning Membrane Binding Geometry of Cytochrome c by Polarized Light Spectroscopy

In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) analysis of the peptide ultraviolet-visible spectrum of a membrane-associated protein (cytochrome (cyt) c) allows orientation and structure to be assessed with quite high accuracy in a native membrane environment that can be systematically varied with respect to lipid composition. Cyt c binds strongly to negatively charged lipid bilayers with a distinct orientation in which its α-helical segments are on average parallel to the membrane surface. Further information is provided by the LD of the π-π∗ transitions of the heme porphyrin and transitions of aromatic residues, mainly a single tryptophan. A good correlation with NMR data was found, and combining NMR structural data with LD angular data allowed the whole protein to be docked to the lipid membrane. When the redox state of cyt c was changed, distinct variations in the LD spectrum of the heme Soret band were seen corresponding to changes in electronic transition energies; however, no significant change in the overall protein orientation or structure was observed. Cyt c is known to interact in a specific manner with the doubly negatively charged lipid cardiolipin, and incorporation of this lipid into the membrane at physiologically relevant levels was indeed found to affect the protein orientation and its α-helical content. The detail in which cyt c binding is described in this study shows the potential of LD spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.

Potassium Transport in Corynebacterium glutamicum Is Facilitated by the Putative Channel Protein CglK, Which Is Essential for pH Homeostasis and Growth at Acidic pH

We studied the requirement for potassium and for potassium transport activity for the biotechnologically important bacterium Corynebacterium glutamicum, which is used for large-scale production of amino acids. Different from many other bacteria, at alkaline or neutral pH, C. glutamicum is able to grow without the addition of potassium, resulting in very low cytoplasmic potassium concentrations. In contrast, at acidic pH, the ability for growth was found to depend on the presence of K+. For the first time, we provide experimental evidence that a potential potassium channel (CglK) acts as the major potassium uptake system in a bacterium and proved CglK's function directly in its natural membrane environment. A full-length CglK protein and a separate soluble protein harboring the RCK domain can be translated from the cglK gene, and both are essential for full CglK functionality. As a reason for potassium-dependent growth limitation at acidic pH, we identified the impaired capacity for internal pH homeostasis, which depends on the availability and internal accumulation of potassium. Potassium uptake via CglK was found to be relevant for major physiological processes, like the activity of the respiratory chain, and to be crucial for maintenance of the internal pH, as well as for the adjustment of the membrane potential in C. glutamicum.

Rhodopsin Activation Switches in a Native Membrane Environment†

The elucidation of structure-function relationships of membrane proteins still poses a considerable challenge due to the sometimes profound influence of the lipid bilayer on the functional properties of the protein. The visual pigment rhodopsin is a prototype of the family of G protein-coupled transmembrane receptors and a considerable part of our knowledge on its activation mechanisms has been derived from studies on detergent-solubilized proteins. This includes in particular the events associated with the conformational transitions of the receptor from the still inactive Meta I to the Meta II photoproduct states, which are involved in signaling. These events involve disruption of an internal salt bridge of the retinal protonated Schiff base, movement of helices and proton uptake from the solvent by the conserved cytoplasmic E(D)RY network around Glu134. As the equilibria associated with these events are considerably altered by the detergent environment, we set out to investigate these equilibria in the native membrane environment and to develop a coherent thermodynamic model of these activating steps using UV-visible and Fourier-transform infrared spectroscopy as complementary techniques. Particular emphasis is put on the role of protonation of Glu134 from the solvent, which is a thermodynamic prerequisite for full receptor activation in membranes, but not in detergent. In view of the conservation of this carboxylate group in family A G protein-coupled receptors, it may also play a similar role in the activation of other family members.

Influence of Dynamics on The Analysis of Solid-State NMR Data From Membrane-bound Peptides

By isotope labeling of membrane-bound peptides, typically with 2H, 19F, or 15N, solid-state NMR experiments can yield data from which the orientation of peptides in a native membrane environment can be determined. Such an orientation is defined by a tilt angle and an azimuthal rotation angle.Here we show that to obtain correct values of the orientation angles, it is important to include dynamics in the analysis of the NMR data. Nevertheless the effects of dynamics are different depending on the type of isotope labeling and NMR experiment considered.To analyze the influence of dynamics in detail, we generated virtual NMR observables using a model peptide undergoing explicit Gaussian fluctuations of the orientation angles. For simulated 2H- or 19F-NMR data, even moderate motions were found to have a large influence, as calculated tilt values are consistently much too small, unless dynamics is properly considered. A simple dynamic model, including a molecular order parameter scaling factor, gives good results only for moderately mobile peptides, while for high mobility cases the correct tilt is only obtained by re-introducing the explicit Gaussian fluctuations in the fitting functions.In contrast, 15N-NMR data appear to be less sensitive to rigid-body peptide motions, and PISEMA spectra can give correct orientations even for highly mobile peptides, and assuming a static model for the analysis. The differences are due to the different orientation of the tensors of 2H- and 19F-labels, placed on peptide side chains, compared to the orientation of the 15N tensor, placed on amide backbone groups.We conclude that dynamics should be included in the analysis of solid-state NMR data of membrane-bound peptides. Not only does this give more accurate orientations, but it can also provide information about the dynamics of the peptide.

Influence of Proline on the Thermostability of the Active Site and Membrane Arrangement of Transmembrane Proteins

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro 50, Pro 91, and Pro 186 in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro 91, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro 91 and Pro 186 showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.

Computational studies of the binding site of α1A-adrenoceptor antagonists

Aimed at achieving a good understanding of the 3-dimensional structures of human alpha1A-adrenoceptor (alpha1A-AR), we have successfully developed its homology model based on the crystal structure of beta2-AR. Subsequent structural refinements were performed to mimic the receptor's natural membrane environment by using molecular mechanics (MM) and molecular dynamics (MD) simulations in the GBSW implicit membrane model. Through molecular docking and further simulations, possible binding modes of subtype-selective alpha1A-AR antagonists, Silodosin, RWJ-69736 and (+)SNAP-7915, were examined. Results of the modeling and docking studies are qualitatively consistent with available experimental data from mutagenesis studies. The homology model built should be very useful for designing more potent subtype-selective alpha1A-AR antagonists and for guiding further mutagenesis studies.