Biomedical subjects

James D Lear

Publications and source records attributed to James D Lear.

At least 19 recordsLinked to original sources

PubMed2005

Design of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions.

We have designed polypeptides combining selected lipophilic (LP) and hydrophilic (HP) sequences that assemble into amphiphilic (AP) alpha-helical bundles to reproduce key structure characteristics and functional elements of natural membrane proteins. The principal AP maquette (AP1) developed here joins 14 residues of a heme binding sequence from a structured diheme-four-alpha-helical bundle (HP1), with 24 residues of a membrane-spanning LP domain from the natural four-alpha-helical M2 channel of the influenza virus, through a flexible linking sequence (GGNG) to make a 42 amino acid peptide. The individual AP1 helices (without connecting loops) assemble in detergent into four-alpha-helical bundles as observed by analytical ultracentrifugation. The helices are oriented parallel as indicated by interactions typical of adjacent hemes. AP1 orients vectorially at nonpolar-polar interfaces and readily incorporates into phospholipid vesicles with >97% efficiency, although most probably without vectorial bias. Mono- and diheme-AP1 in membranes enhance functional elements well established in related HP analogues. These include strong redox charge coupling of heme with interior glutamates and internal electric field effects eliciting a remarkable 160 mV splitting of the redox potentials of adjacent hemes that leads to differential heme binding affinities. The AP maquette variants, AP2 and AP3, removed heme-ligating histidines from the HP domain and included heme-ligating histidines in LP domains by selecting the b(H) heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc(1). These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.

Binding Sites↗

PubMed2005

Factor XIa dimer in the activation of factor IX.

Factor XI, unlike other coagulation proteins, is a homodimer of two identical subunits linked by a single disulfide bond formed by Cys321. The present study was undertaken to understand the physiological significance of the dimeric nature of factor XI. We have expressed a mutant FXI/G326C in which the Gly326 residue of factor XI has been mutated to Cys326, reasoning that Cys321 would form an intrachain disulfide bond with Cys326 as in prekallikrein, a plasma protein that exists as a monomer even with 58% amino acid sequence identity and a domain structure very similar to factor XI. No free thiol could be detected in the expressed protein, and it migrated as a monomer on nonreduced SDS-PAGE. In physiological buffer, however, the protein was found to exist in a state of monomer-dimer equilibrium as assessed by gel-filtration chromatography and ultracentrifugation studies (K(d) approximately 36 nM). Functional studies revealed that FXI/G326C was indistinguishable from plasma factor XI in a plasma-clotting assay and in a factor IX activation assay both in the presence and absence of activated platelets even at concentrations at which less than 5% of the mutant exists as dimers. We conclude that, for optimal function in the presence of activated platelets, a preformed dimer of factor XI is not required.

Amino Acid Substitution↗

PubMed2005

Synergistic interactions between aqueous and membrane domains of a designed protein determine its fold and stability.

Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which contribute to folding and stability. To explore the interplay between these two domains we have designed and studied the assembly of coiled-coil peptides that span from the membrane into the aqueous phase. The membrane-spanning segment is based on MS1, a transmembrane coiled coil that contains a single Asn at a buried a position of a central heptad in its sequence. This Asn has been shown to drive assembly of the monomeric peptide in a membrane environment to a mixture of dimers and trimers. The coiled coil has now been extended into the aqueous phase by addition of water-soluble helical extensions. Although too short to fold in isolation, these helical extensions were expected to interact synergistically with the transmembrane domain and modulate its stability as well as its conformational specificity for forming dimers versus trimers. One design contains Asn at a position of the aqueous helical extension, which was expected to specify a dimeric state; a second peptide, which contains Val at this position, was expected to form trimers. The thermodynamics of assembly of the hybrid peptides were studied in micelles by sedimentation equilibrium ultracentrifugation. The aqueous helical extensions indeed conferred additional stability and conformational specificity to MS1 in the expected manner. These studies highlight the delicate interplay between membrane-spanning and water-soluble regions of proteins, and demonstrate how these different environments define the thermodynamics of a given specific interaction. In this case, an Asn in the transmembrane domain provided a strong driving force for folding but failed to specify a unique oligomerization state, while an Asn in the water-soluble domain was able to define specificity for a specific aggregation state as well as modulate stability.

Amino Acid Sequence↗

PubMed2005

A push-pull mechanism for regulating integrin function.

Homomeric and heteromeric interactions between the alphaIIb and beta3 transmembrane domains are involved in the regulation of integrin alphaIIbbeta3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of alphaIIbbeta3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the alphaIIb transmembrane domain. One mutation that enhanced the dimerization of the alphaIIb transmembrane domain indeed induced constitutive alphaIIbbeta3 activation. However, a series of mutations that disrupted homodimerization also led to alphaIIbbeta3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the alphaIIb transmembrane domain, and that mutations that disrupt the alphaIIb/beta3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for alphaIIbbeta3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an alphaIIb/beta3 transmembrane domain heterodimer and by stabilizing the resulting alphaIIb and beta3 transmembrane domain homodimers.

Animals↗

PubMed2005

Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function.

The driving forces behind the folding processes of integral membrane proteins after insertion into the bilayer, is currently under debate. The M2 protein from the influenza A virus is an ideal system to study lateral association of transmembrane helices. Its proton selective channel is essential for virus functioning and a target of the drug amantadine. A 25 residue transmembrane fragment of M2, M2TM, forms a four-helix bundle in vivo and in various detergents and phospholipid bilayers. Presented here are the energetic consequences for mutations made to the helix/helix interfaces of the M2TM tetramer. Analytical ultracentrifugation has been used to determine the effect of ten single-site mutations, to either alanine or phenylalanine, on the oligomeric state and the free energy of M2TM in the absence and the presence of amantadine. It was expected that many of these mutations would perturb the M2TM stability and tetrameric integrity. Interestingly, none of the mutations destabilize tetramerization. This finding suggests that M2 sacrifices stability to preserve its functions, which require rapid and specific interchange between distinct conformations involved in gating and proton conduction. Mutations might therefore restrict the full range of conformations by stabilizing a given native or non-native conformational state. In order to assess one specific conformation of the tetramer, we measured the binding of amantadine to the resting state of the channel, and examined the overall free energy of assembly of the amantadine bound tetramer. All of the mutations destabilized amantadine binding or were isoenergetic. We also find that large to small residue changes destabilize the amantadine bound tetramer whereas mutations to side-chains of similar volume stabilize this conformation. A structural model of the amantadine bound state of M2TM was generated using a novel protocol that optimizes a structure for an ensemble of neutral and disruptive mutations. The model structure is consistent with the mutational data.

Amantadine↗

PubMed2005

Relationship between side chain structure and 14-helix stability of beta3-peptides in water.

Folded polymers are used in Nature for virtually every vital process. Nonnatural folded polymers, or foldamers, have the potential for similar versatility, and the design and refinement of such molecules is of considerable current interest. Here we report a complete and systematic analysis of the relationship between side chain structure and the 14-helicity of a well-studied class of foldamers, beta(3)-peptides, in water. Our experimental results (1) verify the importance of macrodipole stabilization for maintaining 14-helix structure, (2) provide comprehensive evidence that beta(3)-amino acids branched at the first side chain carbon are 14-helix-stabilizing, (3) suggest a novel role for side chain hydrogen bonding as an additional stabilizing force in beta(3)-peptides containing beta(3)-homoserine or beta(3)-homothreonine, and (4) demonstrate that diverse functionality can be incorporated into a stable 14-helix. Gas- and solution-phase calculations and Monte Carlo simulations recapitulate the experimental trends only in the context of oligomers, yielding insight into the mechanisms behind 14-helix folding. The 14-helix propensities of beta(3)-amino acids differ starkly from the alpha-helix propensities of analogous alpha-amino acids. This contrast informs current models for alpha-helix folding, and suggests that 14-helix folding is governed by different biophysical forces than is alpha-helix folding. The ability to modulate 14-helix structure through side chain choice will assist rational design of 14-helical beta-peptide ligands for macromolecular targets.

Amino Acids↗

PubMed2004

Volatile anesthetic modulation of oligomerization equilibria in a hexameric model peptide.

To determine if occupancy of interfacial pockets in oligomeric proteins by volatile anesthetic molecules can allosterically regulate oligomerization equilibria, variants of a three-helix bundle peptide able to form higher oligomers were studied with analytical ultracentrifugation, hydrogen exchange and modeling. Halothane shifted the oligomerization equilibria towards the oligomer only in a mutation predicted to create sufficient volume in the hexameric pocket. Other mutations at this residue, predicted to create a too small or too polar pocket, were unaffected by halothane. Inhaled anesthetic modulation of oligomerization interactions is a novel and potentially generalizable biophysical basis for some anesthetic actions.

Allosteric Regulation↗

PubMed2004

Association of a model transmembrane peptide containing gly in a heptad sequence motif.

A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A.

Amino Acid Motifs↗

PubMed2004

Helical beta-peptide inhibitors of the p53-hDM2 interaction.

hDM2 is recognized in vivo by a short alpha-helix within the p53 trans-activation domain (p53AD). Disruption of the p53.hDM2 interaction is an important goal for cancer therapy. A functional epitope comprised of three residues on one face of the p53AD helix (F19, W23, and L26) contributes heavily to the binding free energy. We hypothesized that the p53AD functional epitope would be recapitulated if the side chains of F19, W23, and L26 were presented at successive positions three residues apart on a stabilized beta3-peptide 14-helix. Here, we report a set of beta3-peptides that possess significant 14-helix structure in water; one recognizes a cleft on the surface of hDM2 with nanomolar affinity. The strategy for beta3-peptide design that we describe is general and may have advantages over one in which individual or multiple beta-amino acid substitutions are introduced into a functional alpha-peptide, because it is based on homology at the level of secondary structure, not primary sequence.

Circular Dichroism↗

PubMed2004

Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes.

Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin alpha and beta subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type alpha(IIb) TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type alpha(IIb) TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the alpha(IIb) TM domain oligomerizes in biological membranes. Mutating each of the alpha(IIb) TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of alpha(IIb) TM helix. These data were used to generate an atomic model of the alpha(IIb) homodimer, revealing a family of structures with right-handed crossing angles of 40 degrees to 60 degrees, consistent with a 4.0-residue periodicity, and with an interface rotated by 50 degrees relative to glycophorin A. Thus, although the alpha(IIb) TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.

Amino Acid Motifs↗

PubMed2004

Computational design of water-soluble analogues of the potassium channel KcsA.

Although the interiors of membrane and water-soluble proteins are similar in their physicochemical properties, membrane proteins differ in having larger fractions of hydrophobic residues on their exteriors. Thus, it should be possible to water-solubilize membrane proteins by mutating their lipid-contacting side chains to more polar groups. Here, a computational approach was used to generate water-soluble variants of the potassium channel KcsA. As a probe of the correctness of the fold, the proteins contain an agitoxin2 binding site from a mammalian homologue of the channel. The resulting proteins express in high yield in Escherichia coli and share the intended functional and structural properties with KcsA, including secondary structure, tetrameric quaternary structure, and tight specific binding to both agitoxin2 and a small molecule channel blocker.

Amino Acid Sequence↗

PubMed2004

Oligomerization of fusogenic peptides promotes membrane fusion by enhancing membrane destabilization.

A key element of membrane fusion reactions in biology is the involvement of specific fusion proteins. In many viruses, the proteins that mediate membrane fusion usually exist as homotrimers. Furthermore, they contain extended triple-helical coiled-coil domains and fusogenic peptides. It has been suggested that the coiled-coil domains present the fusogenic peptide in a conformation or geometry favorable for membrane fusion. To test the hypothesis that trimerization of fusogenic peptide is related to optimal fusion, we have designed and synthesized a triple-stranded coiled-coil X31 peptide, also known as the ccX31, which mimics the influenza virus hemagglutinin fusion peptide in the fusion-active state. We compared the membrane interactive properties of ccX31 versus the monomeric X31 fusogenic peptide. Our data show that trimerization enhances peptide-induced leakage of liposomal contents and lipid mixing. Furthermore, studies using micropipette aspiration of single vesicles reveal that ccX31 decreases lysis tension, tau(lysis), but not area expansion modulus, Ka, of phospholipid bilayers, whereas monomeric X31 peptide lowers both tau(lysis) and Ka. Our results are consistent with the hypothesis that oligomerization of fusogenic peptide promotes membrane fusion, possibly by enhancing localized destabilization of lipid bilayers.

Dimerization↗

PubMed2003

Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers.

Despite significant efforts and promising progress, the understanding of membrane protein folding lags behind that of soluble proteins. Insights into the energetics of membrane protein folding have been gained from biophysical studies in membrane-mimicking environments (primarily detergent micelles). However, the development of techniques for studying the thermodynamics of folding in phospholipid bilayers remains a considerable challenge. We had previously used thiol-disulfide exchange to study the thermodynamics of association of transmembrane alpha-helices in detergent micelles; here, we extend this methodology to phospholipid bilayers. The system for this study is the homotetrameric M2 proton channel protein from the influenza A virus. Transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. Thiol-disulfide exchange under equilibrium conditions was used to quantitatively measure the thermodynamics of this folding interaction in phospholipid bilayers. The effects of phospholipid acyl chain length and cholesterol on the peptide association were investigated. The association of the helices strongly depends on the thickness of the bilayer and cholesterol levels present in the phospholipid bilayer. The most favorable folding occurred when there was a good match between the width of the apolar region of the bilayer and the hydrophobic length of the transmembrane helix. Physiologically relevant variations in the cholesterol level are sufficient to strongly influence the association. Evaluation of the energetics of peptide association in the presence and absence of cholesterol showed a significantly tighter association upon inclusion of cholesterol in the lipid bilayers.

Biophysical Phenomena↗

PubMed2003

Proton conduction through the M2 protein of the influenza A virus; a quantitative, mechanistic analysis of experimental data.

The M2 proton channel from influenza A virus forms proton-selective ion channels, which are the target of the drug amantadine. Here, existing experimental data are quantitatively examined for insights into mechanisms to account for the pH- and voltage-dependences of M2 proton conduction. The analysis shows that a model involving protonation equilibria of His37, including pH-dependent changes in the relative rates of diffusion on either side of the pore, is quantitatively able to account for recently reported electrophysiological data examining the pH- and voltage-dependences of Rostock and Weybridge strain M2 proton conduction.

Algorithms↗

PubMed2003

Direct analysis of protein sedimentation equilibrium in detergent solutions without density matching.

Characterizing membrane proteins by sedimentation equilibrium is challenging because detergents and/or lipid molecules, usually required for solubilization, form a complex with the protein. The most common way to overcome this problem is Tanford and Reynolds' density matching method, which eliminates the buoyant mass contributions of detergents/lipids by adjusting the solvent density with D2O/H2O mixtures to render either detergent or lipid molecules neutrally buoyant. Unfortunately, the method is practical only for detergent densities between 1.0 (H2O) and 1.1 (D2O) g ml(-1), excluding many of the more commonly used detergents for membrane protein studies. Here, we present a modern variant of Tanford and Reynolds' method that (1) is applicable to any detergent regardless of its specific density, (2) does not compromise accuracy and precision, and (3) provides additional information about the number of detergent molecules that are bound to each protein. The new method was applied successfully to Delta(1-43)A-I, an amino-terminal deletion mutant of human apolipoprotein A-I. Interestingly, we observed a significantly lower Delta(1-43)A-I/octyl-glucoside complex partial specific volume than that expected from volume additivity rules, indicative of specific protein-detergent interactions.

Apolipoprotein A-I↗

PubMed2003

Position-dependence of stabilizing polar interactions of asparagine in transmembrane helical bundles.

Recent studies with model peptides and statistical analyses of the crystal structures of membrane proteins have shown that buried polar interactions contribute significantly to the stabilization of the three-dimensional structures of membrane proteins. Here, we probe how the location of these polar groups along the transmembrane helices affect their free energies of interaction. Asn residues were placed singly and in pairs at three positions within a model transmembrane helix, which had previously been shown to support the formation of trimers in micelles. The model helix was designed to form a transmembrane coiled coil, with Val side chains at the "a" positions of the heptad repeat. Variants of this peptide were prepared in which an Asn residue was introduced at one or more of the "a" positions, and their free energies of association were determined by analytical ultracentrifugation. When placed near the middle of the transmembrane helix, the formation of trimers was stabilized by at least -2.0 kcal/mol per Asn side chain. When the Asn was placed at the interface between the hydrophobic and polar regions of the peptide, the substitution was neither stabilizing nor destabilizing (0.0 +/- 0.5 kcal/mol of monomer). Finally, it has previously been shown that a Val-for-Asn mutation in a water-soluble coiled coil destabilizes the structure by approximately 1.5 kcal/mol of monomer [Acharya, A., et al. (2002) Biochemistry 41, 14122-14131]. Thus, the headgroup region of a micelle appears to have a conformational impact intermediate between that of bulk water and the apolar region of micelle. A similarly large dependence on the location of the polar residues was found in a statistical survey of helical transmembrane proteins. The tendency of different types of residues to be buried in the interiors versus being exposed to lipids was analyzed. Asn and Gln show a very strong tendency to be buried when they are located near the middle of a transmembrane helix. However, when placed near the ends of transmembrane helices, they show little preference for the surface versus the interior of the protein. These data show that Asn side chains within the apolar region of the transmembrane helix provide a significantly larger driving force for association than Asn residues near the apolar/polar interface. Thus, although polar interactions are able to strongly stabilize the folding of membrane proteins, the energetics of association depend on their location within the hydrophobic region of a transmembrane helix.

Amino Acid Sequence↗

PubMed2003

Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations.

Transmembrane helices of integrin alpha and beta subunits have been implicated in the regulation of integrin activity. Two mutations, glycine-708 to asparagine-708 (G708N)and methionine-701 to asparagine-701, in the transmembrane helix of the beta3 subunit enabled integrin alphaIIbbeta3 to constitutively bind soluble fibrinogen. Further characterization of the G708N mutant revealed that it induced alphaIIbbeta3 clustering and constitutive phosphorylation of focal adhesion kinase. This mutation also enhanced the tendency of the transmembrane helix to form homotrimers. These results suggest that homomeric associations involving transmembrane domains provide a driving force for integrin activation. They also suggest a structural basis for the coincidence of integrin activation and clustering.

Amino Acid Sequence↗

PubMed2003

Computational design of a water-soluble analog of phospholamban.

Membrane proteins and water-soluble proteins share a similar core. This similarity suggests that it should be possible to water-solubilize membrane proteins by mutating only their lipid-exposed residues. We have developed computational tools to design water-soluble variants of helical membrane proteins, using the pentameric phospholamban (PLB) as our test case. To water-solublize PLB, the membrane-exposed positions were changed to polar or charged amino acids, while the putative core was left unaltered. We generated water-soluble phospholamban (WSPLB), and compared its properties to its predecessor PLB. In aqueous solution, WSPLB mimics all of the reported properties of PLB including oligomerization state, helical structure, and stabilization upon phosphorylation. We also characterized the truncated mutant WSPLB (21-52) comprising only the former transmembrane segment of PLB. This peptide shows a decreased specificity for forming a pentameric oligomerization state.

Algorithms↗