Simon Newstead - Academia.edu (original) (raw)

Papers by Simon Newstead

Research paper thumbnail of MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes

Structure (London, England : 1993), Jan 28, 2015

There has been exponential growth in the number of membrane protein structures determined. Nevert... more There has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interaction...

Research paper thumbnail of Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Biophysical journal, Jan 24, 2015

Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also inv... more Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1-S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it pr...

Research paper thumbnail of Galactose recognition by the carbohydrate-binding module of a bacterial sialidase

Acta crystallographica. Section D, Biological crystallography, 2005

Glycoside hydrolases often possess carbohydrate-binding modules (CBMs) in addition to their catal... more Glycoside hydrolases often possess carbohydrate-binding modules (CBMs) in addition to their catalytic domains, which help target the enzymes to appropriate substrates and thereby increase their catalytic efficiency. Sialidases hydrolyse the release of sialic acid from a variety of glycoconjugates and play significant roles in the pathogenesis of a number of important diseases. The sialidase from Micromonospora viridifaciens has a CBM which recognizes galactose. The CBM is linked to the catalytic domain by an immunoglobulin-like domain, resulting in the galactose binding site sitting above the catalytic site, suggesting an interplay between the two sites. By studying nine crystallographically independent structures of the M. viridifaciens sialidase, the relative flexibility of the three domains was analysed. A detailed study is also presented of the recognition of galactose and lactose by the M. viridifaciens CBM. The striking structure of this sialidase suggests a role for the CBM i...

Research paper thumbnail of Structure and mechanism of action of an inverting mutant sialidase

Biochemistry, Jan 28, 2005

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to sma... more Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Brønsted parameters (beta(lg)) on k(cat) and k(cat)/K(m) are -0.63 +/- 0.05 and -0.80 +/- 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium alpha-d-N-acetylneuraminide (DHP-alphaNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via S(N)1 and S(N)2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported...

Research paper thumbnail of Thermodynamic evidence for a dual transport mechanism in a POT peptide transporter

Research paper thumbnail of The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates

The Journal of biological chemistry, Jan 4, 2008

Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, an... more Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97A resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97A resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5A resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2A resolution. Together,...

Research paper thumbnail of Crystal Structure of Fluc, a Microbial Fluoride Channel

Biophysical Journal, 2015

Research paper thumbnail of Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters

EMBO reports, 2014

An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as ex... more An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochemically distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.

Research paper thumbnail of Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

Research paper thumbnail of Benchmarking Membrane Protein Detergent Stability for Improving Throughput of High-Resolution X-ray Structures

Structure, 2011

Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane pr... more Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield highresolution structures.

Research paper thumbnail of Insights into How Nucleotide-Binding Domains Power ABC Transport

Research paper thumbnail of A high-throughput method for membrane protein solubility screening: The ultracentrifugation dispersity sedimentation assay

Protein Science, 2007

One key to successful crystallization of membrane proteins is the identification of detergents th... more One key to successful crystallization of membrane proteins is the identification of detergents that maintain the protein in a soluble, monodispersed state. Because of their hydrophobic nature, membrane proteins are particularly prone to forming insoluble aggregates over time. This nonspecific aggregation of the molecules reduces the likelihood of the regular association of the protein molecules essential for crystal lattice formation. Critical buffer components affecting the aggregation of membrane proteins include detergent choice, salt concentration, and presence of glycerol. The optimization of these parameters is often a time-and protein-consuming process. Here we describe a novel ultracentrifugation dispersity sedimentation (UDS) assay in which ultracentrifugation of very small (5 mL) volumes of purified, soluble membrane protein is combined with SDS-PAGE analysis to rapidly assess the degree of protein aggregation. The results from the UDS method correlate very well with established methods like size-exclusion chromatography (SEC), while consuming considerably less protein. In addition, the UDS method allows rapid screening of detergents for membrane protein crystallization in a fraction of the time required by SEC. Here we use the UDS method in the identification of suitable detergents and buffer compositions for the crystallization of three recombinant prokaryotic membrane proteins. The implications of our results for membrane protein crystallization prescreening are discussed.

Research paper thumbnail of Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones

Protein Science, 2009

Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate al... more Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t 1/2 Ӎ 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca 2+ , Mg 2+ , and Mn 2+ can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg 2+ can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39°C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.

Research paper thumbnail of Rationalizing α-helical membrane protein crystallization

Protein Science, 2008

X-ray crystallography is currently the most successful method for determining the three-dimension... more X-ray crystallography is currently the most successful method for determining the three-dimensional structure of membrane proteins. Nevertheless, growing the crystals required for this technique presents one of the major bottlenecks in this area of structural biology. This is especially true for the alpha-helical type membrane proteins that are of particular interest due to their medical relevance. To address this problem we have undertaken a detailed analysis of the crystallization conditions from 121 alpha-helical membrane protein structures deposited in the Protein Data Bank. This information has been analyzed so that the success of different parameters can be easily compared for different membrane protein families. Concurrent with this analysis, we also present the new sparse matrix crystallization screen MemGold.

Research paper thumbnail of High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences, 2007

Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a signi... more Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Based on the analysis of 43 eukaryotic membrane proteins, we present a cost-effective highthroughput approach for rapidly screening membrane proteins that can be overproduced to levels of >1 mg per liter in Saccharomyces cerevisiae. We find that 70% of the well expressed membrane proteins tested in this system are stable, targeted to the correct organelle, and monodisperse in either Fos-choline 12 (FC-12) or n-dodecyl-␤-D-maltoside. We illustrate the advantage of such an approach, with the purification of monodisperse human and yeast nucleotide-sugar transporters to unprecedented levels.

Research paper thumbnail of GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

Research paper thumbnail of Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1

Nature, 2014

The NRT1/PTR family of proton-coupled transporters are responsible for nitrogen assimilation in e... more The NRT1/PTR family of proton-coupled transporters are responsible for nitrogen assimilation in eukaryotes and bacteria through the uptake of peptides. However, in most plant species members of this family have evolved to transport nitrate as well as additional secondary metabolites and hormones. In response to falling nitrate levels, NRT1.1 is phosphorylated on an intracellular threonine that switches the transporter from a low-affinity to high-affinity state. Here we present both the apo and nitrate-bound crystal structures of Arabidopsis thaliana NRT1.1, which together with in vitro binding and transport data identify a key role for His 356 in nitrate binding. Our data support a model whereby phosphorylation increases structural flexibility and in turn the rate of transport. Comparison with peptide transporters further reveals how the NRT1/PTR family has evolved to recognize diverse nitrogenous ligands, while maintaining elements of a conserved coupling mechanism within this superfamily of nutrient transporters.

Research paper thumbnail of The structure of the efflux pump AcrB in complex with bile acid

Molecular Membrane Biology, 2008

Gastrointestinal bacteria, like Escherichia coli, must remove bile acid to survive in the gut. Bi... more Gastrointestinal bacteria, like Escherichia coli, must remove bile acid to survive in the gut. Bile acid removal in E. coli is thought to be mediated primarily by the multidrug efflux pump, AcrB. Here, we present the structure of E. coli AcrB in complex with deoxycholate at 3.85 A resolution. All evidence suggests that bile acid is transported out of the cell via the periplasmic vestibule of the AcrAB-TolC complex.

Research paper thumbnail of Insights into outer membrane protein crystallization

Molecular Membrane Biology, 2008

Outer membrane proteins are structurally distinct from those that reside in the inner membrane an... more Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on > 40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either β-sheets or less commonly from α-helices. The most common architecture is the β-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallisation can be made. Herein we analyse the crystallisation data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallisation is presented based on the present analysis.

Research paper thumbnail of Contribution of the active site aspartic acid to catalysis in the bacterial neuraminidase from Micromonospora viridifaciens

FEBS Letters, 2004

A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed ... more A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed and purified. Kinetic studies reveal that the replacement of the conserved aspartic acid with glycine results in a catalytically competent retaining sialidase that possesses significant activity against activated substrates. The contribution of this aspartate residue to the free energy of hydrolysis for natural substrates is greater than 19 kJ/mol. The three dimensional structure of the D92G mutant shows that the removal of aspartic acid 92 causes no significant re-arrangement of the active site, and that an ordered water molecule substitutes for the carboxylate group of D92.

Research paper thumbnail of MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes

Structure (London, England : 1993), Jan 28, 2015

There has been exponential growth in the number of membrane protein structures determined. Nevert... more There has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interaction...

Research paper thumbnail of Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Biophysical journal, Jan 24, 2015

Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also inv... more Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1-S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it pr...

Research paper thumbnail of Galactose recognition by the carbohydrate-binding module of a bacterial sialidase

Acta crystallographica. Section D, Biological crystallography, 2005

Glycoside hydrolases often possess carbohydrate-binding modules (CBMs) in addition to their catal... more Glycoside hydrolases often possess carbohydrate-binding modules (CBMs) in addition to their catalytic domains, which help target the enzymes to appropriate substrates and thereby increase their catalytic efficiency. Sialidases hydrolyse the release of sialic acid from a variety of glycoconjugates and play significant roles in the pathogenesis of a number of important diseases. The sialidase from Micromonospora viridifaciens has a CBM which recognizes galactose. The CBM is linked to the catalytic domain by an immunoglobulin-like domain, resulting in the galactose binding site sitting above the catalytic site, suggesting an interplay between the two sites. By studying nine crystallographically independent structures of the M. viridifaciens sialidase, the relative flexibility of the three domains was analysed. A detailed study is also presented of the recognition of galactose and lactose by the M. viridifaciens CBM. The striking structure of this sialidase suggests a role for the CBM i...

Research paper thumbnail of Structure and mechanism of action of an inverting mutant sialidase

Biochemistry, Jan 28, 2005

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to sma... more Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Brønsted parameters (beta(lg)) on k(cat) and k(cat)/K(m) are -0.63 +/- 0.05 and -0.80 +/- 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium alpha-d-N-acetylneuraminide (DHP-alphaNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via S(N)1 and S(N)2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported...

Research paper thumbnail of Thermodynamic evidence for a dual transport mechanism in a POT peptide transporter

Research paper thumbnail of The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates

The Journal of biological chemistry, Jan 4, 2008

Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, an... more Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97A resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97A resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5A resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2A resolution. Together,...

Research paper thumbnail of Crystal Structure of Fluc, a Microbial Fluoride Channel

Biophysical Journal, 2015

Research paper thumbnail of Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters

EMBO reports, 2014

An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as ex... more An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochemically distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.

Research paper thumbnail of Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

Research paper thumbnail of Benchmarking Membrane Protein Detergent Stability for Improving Throughput of High-Resolution X-ray Structures

Structure, 2011

Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane pr... more Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield highresolution structures.

Research paper thumbnail of Insights into How Nucleotide-Binding Domains Power ABC Transport

Research paper thumbnail of A high-throughput method for membrane protein solubility screening: The ultracentrifugation dispersity sedimentation assay

Protein Science, 2007

One key to successful crystallization of membrane proteins is the identification of detergents th... more One key to successful crystallization of membrane proteins is the identification of detergents that maintain the protein in a soluble, monodispersed state. Because of their hydrophobic nature, membrane proteins are particularly prone to forming insoluble aggregates over time. This nonspecific aggregation of the molecules reduces the likelihood of the regular association of the protein molecules essential for crystal lattice formation. Critical buffer components affecting the aggregation of membrane proteins include detergent choice, salt concentration, and presence of glycerol. The optimization of these parameters is often a time-and protein-consuming process. Here we describe a novel ultracentrifugation dispersity sedimentation (UDS) assay in which ultracentrifugation of very small (5 mL) volumes of purified, soluble membrane protein is combined with SDS-PAGE analysis to rapidly assess the degree of protein aggregation. The results from the UDS method correlate very well with established methods like size-exclusion chromatography (SEC), while consuming considerably less protein. In addition, the UDS method allows rapid screening of detergents for membrane protein crystallization in a fraction of the time required by SEC. Here we use the UDS method in the identification of suitable detergents and buffer compositions for the crystallization of three recombinant prokaryotic membrane proteins. The implications of our results for membrane protein crystallization prescreening are discussed.

Research paper thumbnail of Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones

Protein Science, 2009

Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate al... more Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t 1/2 Ӎ 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca 2+ , Mg 2+ , and Mn 2+ can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg 2+ can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39°C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.

Research paper thumbnail of Rationalizing α-helical membrane protein crystallization

Protein Science, 2008

X-ray crystallography is currently the most successful method for determining the three-dimension... more X-ray crystallography is currently the most successful method for determining the three-dimensional structure of membrane proteins. Nevertheless, growing the crystals required for this technique presents one of the major bottlenecks in this area of structural biology. This is especially true for the alpha-helical type membrane proteins that are of particular interest due to their medical relevance. To address this problem we have undertaken a detailed analysis of the crystallization conditions from 121 alpha-helical membrane protein structures deposited in the Protein Data Bank. This information has been analyzed so that the success of different parameters can be easily compared for different membrane protein families. Concurrent with this analysis, we also present the new sparse matrix crystallization screen MemGold.

Research paper thumbnail of High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences, 2007

Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a signi... more Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Based on the analysis of 43 eukaryotic membrane proteins, we present a cost-effective highthroughput approach for rapidly screening membrane proteins that can be overproduced to levels of >1 mg per liter in Saccharomyces cerevisiae. We find that 70% of the well expressed membrane proteins tested in this system are stable, targeted to the correct organelle, and monodisperse in either Fos-choline 12 (FC-12) or n-dodecyl-␤-D-maltoside. We illustrate the advantage of such an approach, with the purification of monodisperse human and yeast nucleotide-sugar transporters to unprecedented levels.

Research paper thumbnail of GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

Research paper thumbnail of Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1

Nature, 2014

The NRT1/PTR family of proton-coupled transporters are responsible for nitrogen assimilation in e... more The NRT1/PTR family of proton-coupled transporters are responsible for nitrogen assimilation in eukaryotes and bacteria through the uptake of peptides. However, in most plant species members of this family have evolved to transport nitrate as well as additional secondary metabolites and hormones. In response to falling nitrate levels, NRT1.1 is phosphorylated on an intracellular threonine that switches the transporter from a low-affinity to high-affinity state. Here we present both the apo and nitrate-bound crystal structures of Arabidopsis thaliana NRT1.1, which together with in vitro binding and transport data identify a key role for His 356 in nitrate binding. Our data support a model whereby phosphorylation increases structural flexibility and in turn the rate of transport. Comparison with peptide transporters further reveals how the NRT1/PTR family has evolved to recognize diverse nitrogenous ligands, while maintaining elements of a conserved coupling mechanism within this superfamily of nutrient transporters.

Research paper thumbnail of The structure of the efflux pump AcrB in complex with bile acid

Molecular Membrane Biology, 2008

Gastrointestinal bacteria, like Escherichia coli, must remove bile acid to survive in the gut. Bi... more Gastrointestinal bacteria, like Escherichia coli, must remove bile acid to survive in the gut. Bile acid removal in E. coli is thought to be mediated primarily by the multidrug efflux pump, AcrB. Here, we present the structure of E. coli AcrB in complex with deoxycholate at 3.85 A resolution. All evidence suggests that bile acid is transported out of the cell via the periplasmic vestibule of the AcrAB-TolC complex.

Research paper thumbnail of Insights into outer membrane protein crystallization

Molecular Membrane Biology, 2008

Outer membrane proteins are structurally distinct from those that reside in the inner membrane an... more Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on > 40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either β-sheets or less commonly from α-helices. The most common architecture is the β-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallisation can be made. Herein we analyse the crystallisation data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallisation is presented based on the present analysis.

Research paper thumbnail of Contribution of the active site aspartic acid to catalysis in the bacterial neuraminidase from Micromonospora viridifaciens

FEBS Letters, 2004

A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed ... more A recombinant D92G mutant sialidase from Micromonospora viridifaciens has been cloned, expressed and purified. Kinetic studies reveal that the replacement of the conserved aspartic acid with glycine results in a catalytically competent retaining sialidase that possesses significant activity against activated substrates. The contribution of this aspartate residue to the free energy of hydrolysis for natural substrates is greater than 19 kJ/mol. The three dimensional structure of the D92G mutant shows that the removal of aspartic acid 92 causes no significant re-arrangement of the active site, and that an ordered water molecule substitutes for the carboxylate group of D92.