- School of Biological Sciences
The University of Auckland
Auckland, New Zealand - +64 9 373 7599
Richard Kingston
The University of Auckland, School of Biological Sciences, Faculty Member
- Structural Biology, Virology, Single crystal x-ray diffraction, Calorimetry, NMR Spectroscopy, Protein crystallography, and 4 moreTransmission Electron Microscopy, CD Spectroscopy, Biophysical Chemistry, and Laser Light Scattering(Transmission Electron Microscopy, CD Spectroscopy, Biophysical Chemistry, and Laser Light Scattering)edit
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The association and dissociation of protein oligomers is frequently coupled to the binding of ligands, facilitating the regulation of many biological processes. Equilibrium thermodynamic models are needed to describe the linkage between... more
The association and dissociation of protein oligomers is frequently coupled to the binding of ligands, facilitating the regulation of many biological processes. Equilibrium thermodynamic models are needed to describe the linkage between ligand binding and homo-oligomerization. These models must be parameterized in a way that makes physical interpretation straightforward, and allows elaborations or simplifications to be readily incorporated. We propose a systematic framework for the equilibrium analysis of ligand-linked oligomerization, treating in detail the case of a homo-oligomer with cyclic point group symmetry, where each subunit binds a ligand at a single site. Exploiting the symmetry of the oligomer, in combination with a nearest-neighbors approximation, we derive a class of site-specific ligand binding models involving only four parameters, irrespective of the size of the oligomer. The model parameters allow direct quantitative assessment of ligand binding cooperativity, and ...
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A procedure is described for direct phase determination in protein crystallography, applicable to crystals with high solvent content. The procedure requires only the diffraction data and an estimate of the solvent content as input. Direct... more
A procedure is described for direct phase determination in protein crystallography, applicable to crystals with high solvent content. The procedure requires only the diffraction data and an estimate of the solvent content as input. Direct phase determination is treated as a constraint satisfaction problem, in which an image is sought that is consistent with both the diffraction data and generic constraints on the density distribution in the crystal. The problem is solved using an iterative projection algorithm, the Difference Map algorithm, which has good global convergence properties, and can locate the correct solution without any initial phase information. Computational efficiency is improved by breaking the problem down into two stages; initial approximation of the molecular envelope at low resolution, followed by subsequent phase determination using all of the data. The molecular envelope is continually updated during the phase determination step. At both stages, the algorithm ...
During a proteolytically-driven maturation process, the ortho-retroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a... more
During a proteolytically-driven maturation process, the ortho-retroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assembles in vitro at physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is p...
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The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly, and stability are still being investigated. The N-terminal and C-terminal domains of CA (NTD and CTD, respectively) engage in both... more
The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly, and stability are still being investigated. The N-terminal and C-terminal domains of CA (NTD and CTD, respectively) engage in both homotypic and heterotypic interactions to create the capsid. Hexameric turrets formed by the NTD decorate the majority of the capsid surface. We report nearly complete solid-state NMR (ssNMR) resonance assignments of Rous sarcoma virus (RSV) CA, assembled into hexamer tubes that mimic the authentic capsid. The ssNMR assignments show that, upon assembly, large conformational changes occur in loops connecting helices, as well as the short 310 helix initiating the CTD. The interdomain linker becomes statically disordered. Combining constraints from ssNMR and cryo-electron microscopy (cryo-EM), we establish an atomic resolution model of the RSV CA tubular assembly using molecular dynamics flexible fitting (MDFF) simulations. On the basis of comparison of...
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At a transcriptional pause site, RNA polymerase (RNAP) takes significantly longer than average to transcribe the nucleotide before moving on to the next position. At the single-molecule level this process is stochastic, while at the... more
At a transcriptional pause site, RNA polymerase (RNAP) takes significantly longer than average to transcribe the nucleotide before moving on to the next position. At the single-molecule level this process is stochastic, while at the ensemble level it plays a variety of important roles in biological systems. The pause signal is complex and invokes interplay between a range of mechanisms. Among these factors are: non-canonical transcription events - such as backtracking and hypertranslocation; the catalytically inactive intermediate state hypothesised to act as a precursor to backtracking; the energetic configuration of basepairing within the DNA/RNA hybrid and of those flanking the transcription bubble; and the structure of the nascent mRNA. There are a variety of plausible models and hypotheses but it is unclear which explanations are better. We performed a systematic comparison of 128 kinetic models of transcription using approximate Bayesian computation. Under this Bayesian framew...
Research Interests: Biology()
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The structure of apo-azurin from Alcaligenes denitrificans has been determined at high resolution by X-ray crystallography. Two separate structure analyses have been carried out, (i) on crystals obtained from solutions of apo-azurin and... more
The structure of apo-azurin from Alcaligenes denitrificans has been determined at high resolution by X-ray crystallography. Two separate structure analyses have been carried out, (i) on crystals obtained from solutions of apo-azurin and (ii) on crystals obtained by removal of copper from previously formed crystals of holo-azurin. Data to 1.8 A resolution were collected from the apo-azurin crystals, by Weissenberg photography (with image plates) using synchrotron radiation and by diffractometry, and the structure was refined by restrained least-squares methods to a final R value of 0.160 for all data in the range 10.0-1.8 A. The final model of 1954 protein atoms, 246 water molecules (66 half-weighted), four SO(4)(2-) ions, and two low-occupancy (0.13 and 0.15) Cu atoms has r.m.s. deviations of 0.012, 0.045 and 0.013 A from standard bond lengths, angle distances and planar groups. For copper-removed azurin, data to 2.2 A were collected by diffractometry and the structure refined by restrained least squares to a final R value of 0.158 for all data in the range 10.0-2.2 A. The final model of 1954 protein atoms, 264 water molecules, two SO(4)(2-) ions, two low occupancy (0.18 and 0.22) metal atoms and one unidentified atom (modelled as S) has r.m.s. deviations of 0.013, 0.047 and 0.012 A from standard bond lengths, angle distances and planar groups. The two structures are essentially identical to each other and show no significant differences from the oxidized and reduced holo-azurin structures. The ligand side chains move slightly closer together following the removal of copper, with the radius of the cavity between the three strongly binding ligands, His 46, His 117 and Cys 112, shrinking from 1.31 A in reduced azurin to 1.24 A in oxidized azurin and 1.16 A in apo-azurin. There is a suggestion of increased flexibility in one of the copper-binding loops but the structure supports the view that the copper site found in holo-azurin is a stable structure, defined by the constraints of the polypeptide structure even in the absence of a bound metal ion.
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Iterative projection algorithms are proposed as a tool for ab initio phasing in virus crystallography. The good global convergence properties of these algorithms, coupled with the spherical shape and high structural redundancy of... more
Iterative projection algorithms are proposed as a tool for ab initio phasing in virus crystallography. The good global convergence properties of these algorithms, coupled with the spherical shape and high structural redundancy of icosahedral viruses, allows high resolution phases to be determined with no initial phase information. This approach is demonstrated by determining the electron density of a virus crystal with 5-fold non-crystallographic symmetry, starting with only a spherical shell envelope. The electron density obtained is sufficiently accurate for model building. The results indicate that iterative projection algorithms should be routinely applicable in virus crystallography, without the need for ancillary phase information.
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Iterative projection algorithms (IPAs) are a promising tool for protein crystallographic phase determination. Although related to traditional density-modification algorithms, IPAs have better convergence properties, and, as a result, can... more
Iterative projection algorithms (IPAs) are a promising tool for protein crystallographic phase determination. Although related to traditional density-modification algorithms, IPAs have better convergence properties, and, as a result, can effectively overcome the phase problem given modest levels of structural redundancy. This is illustrated by applying IPAs to determine the electron densities of two protein crystals with fourfold non-crystallographic symmetry, starting with only the experimental diffraction amplitudes, a low-resolution molecular envelope and the position of the non-crystallographic axes. The algorithm returns electron densities that are sufficiently accurate for model building, allowing automated recovery of the known structures. This study indicates that IPAs should find routine application in protein crystallography, being capable of reconstructing electron densities starting with very little initial phase information.
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Myxovirus resistance (Mx) proteins restrict replication of numerous viruses. They are closely related to membrane-remodeling fission GTPases, such as dynamin. Mx proteins can tubulate lipids and form rings or filaments that may interact... more
Myxovirus resistance (Mx) proteins restrict replication of numerous viruses. They are closely related to membrane-remodeling fission GTPases, such as dynamin. Mx proteins can tubulate lipids and form rings or filaments that may interact directly with viral structures. GTPase domain dimerization is thought to allow crosstalk between the rungs of a tubular or helical assembly, facilitating constriction. We demonstrate that the GTPase domain of MxA dimerizes to facilitate catalysis, in a fashion analogous to dynamin. GTP binding is associated with the lever-like movement of structures adjacent to the GTPase domain, while GTP hydrolysis returns MxA to its resting state. Dimerization is not significantly promoted by substrate binding and occurs only transiently, yet is central to catalytic efficiency. Therefore, we suggest dimerization functions to coordinate the activity of spatially adjacent Mx molecules within an assembly, allowing their mechanical power strokes to be synchronized at ...
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The production and analysis of individual structural domains is a common strategy for studying large or complex proteins, which may be experimentally intractable in their full-length form. However, identifying domain boundaries is... more
The production and analysis of individual structural domains is a common strategy for studying large or complex proteins, which may be experimentally intractable in their full-length form. However, identifying domain boundaries is challenging if there is little structural information concerning the protein target. One experimental procedure for mapping domains is to screen a library of random protein fragments for solubility, since truncation of a domain will typically expose hydrophobic groups, leading to poor fragment solubility. We have coupled fragment solubility screening with global data analysis to develop an effective method for identifying structural domains within a protein. A gene fragment library is generated using mechanical shearing, or by uracil doping of the gene and a uracil-specific enzymatic digest. A split green fluorescent protein (GFP) assay is used to screen the corresponding protein fragments for solubility when expressed in Escherichia coli. The soluble frag...
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Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is... more
Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.
Research Interests: Protein Folding, Protein Science, Magnetic Resonance Spectroscopy, Macromolecular X-Ray Crystallography, Protein Stability, and 9 morePhylogeny, Intrinsically disordered proteins, Molecular Phylogenetics and Evolution, Mumps Virus, Protein Conformation, Amino Acid Sequence, RNA Dependent RNA Polymerase, Biochemistry and cell biology, and Molecular Sequence Data
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The release of NADH from the enzyme.NADH complexes was rate limiting at 37 degrees, for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase. Marked substrate activation was observed at this temperature as was... more
The release of NADH from the enzyme.NADH complexes was rate limiting at 37 degrees, for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase. Marked substrate activation was observed at this temperature as was activation by p-(chloromercuri)benzoate. Activation of enzymic activity may be of importance in vivo.
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A simple algebraic expression is used to calculate the structure-factor amplitudes of the anomalous scatterers within a protein crystal, using X-ray diffraction measurements made at two wavelengths and given a single type of anomalous... more
A simple algebraic expression is used to calculate the structure-factor amplitudes of the anomalous scatterers within a protein crystal, using X-ray diffraction measurements made at two wavelengths and given a single type of anomalous scatterer within the crystal. The expression is exact and is readily derived from the standard algebraic equations for analyzing multi-wavelength anomalous diffraction data. Evaluation of the expression requires estimates for (lambda)f' and (lambda)f", the real and imaginary components of the anomalous scattering, respectively. When these are not known at the wavelengths in question, a statistical procedure is suggested, applicable when the crystal space group has centrosymmetric projections, which allows the expression to be used regardless. The method may provide a useful alternative to existing procedures when (lambda)f' and (lambda)f" are unknown or when data have been measured at a limited number of wavelengths.
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The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally... more
The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very wea...
Research Interests: Viruses()
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Measles virus has a single-stranded RNA genome that is organized into a helical complex by the viral N protein. The resulting structure is termed the nucleocapsid and is traversed by the viral polymerase during RNA synthesis. The P... more
Measles virus has a single-stranded RNA genome that is organized into a helical complex by the viral N protein. The resulting structure is termed the nucleocapsid and is traversed by the viral polymerase during RNA synthesis. The P protein, the noncatalytic subunit of the polymerase, provides the “legs and feet” that allow the polymerase to walk along its protein-RNA template. The polymerase feet are very simple three-helix bundles, only 50 amino acids in size. Previously, we have shown that these feet grasp the viral N protein during movement by attaching to a short sequence (amino acids 487–503) within the disordered and surface-exposed tail of N, causing it to fold into a helix. The result is a weak-affinity complex with a short lifetime, which would allow the polymerase to take rapid steps forward. The structure of the complex was determined using X-ray crystallography. This simple model of binding was challenged by a paper in this journal, claiming that a downstream sequence in the tail of N (amino acids 517–525) was also critical for the association. Its presence was reported to enhance the overall affinity of the polymerase feet for N by three orders of magnitude. We have, therefore, examined binding of the polymerase foot domain to amino acids 477–525 of N using quantitative biophysical techniques, and compared the results to our previous binding studies, performed using amino acids 477–505 of N. We find no evidence that the sequence downstream of amino acid 505 influences binding, validating the original single-site binding model.
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Research Interests: Thermodynamics, Molecular Biology, Protein Folding, NMR Spectroscopy, Crystallization, and 20 moreMacromolecular X-Ray Crystallography, Molecular, Circular Dichroism, Differential scanning calorimetry, Light, DSC, Dynamic Light Scattering, Molten Globule, Protein Complex Detection, Protein Domains, Secondary Structure, Mumps Virus, Circular Dichroism Spectroscopy, HSQC, Recombinant Proteins, DLS, X Ray Crystallography, Static Electricity, Biochemistry and cell biology, and Nucleocapsid(Macromolecular X-Ray Crystallography, Molecular, Circular Dichroism, Differential scanning calorimetry, Light, DSC, Dynamic Light Scattering, Molten Globule, Protein Complex Detection, Protein Domains, Secondary Structure, Mumps Virus, Circular Dichroism Spectroscopy, HSQC, Recombinant Proteins, DLS, X Ray Crystallography, Static Electricity, Biochemistry and cell biology, and Nucleocapsid)
(Macromolecular X-Ray Crystallography, Molecular, Circular Dichroism, Differential scanning calorimetry, Light, DSC, Dynamic Light Scattering, Molten Globule, Protein Complex Detection, Protein Domains, Secondary Structure, Mumps Virus, Circular Dichroism Spectroscopy, HSQC, Recombinant Proteins, DLS, X Ray Crystallography, Static Electricity, Biochemistry and cell biology, and Nucleocapsid)
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Abstract The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a... more
Abstract The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a small, trihelical binding domain at ...
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Research Interests: Virology, Transmission Electron Microscopy, Magnetic Resonance Spectroscopy, Early Childhood, Measles Virus, and 12 moreBiological Sciences, Humans, Circular Dichroism, Escherichia coli, Mumps Virus, Isothermal Titration Calorimetry, Calorimetry, Protein Binding, Binding Site, Dissociation Constant, Nucleotides, and Nucleocapsid
We have studied the organization of mature infectious Rous sarcoma virus (RSV), suspended in vitreous ice, using transmission electron microscopy. The enveloped virions are spherical in shape, have a mean diameter of 127 nm, and vary... more
We have studied the organization of mature infectious Rous sarcoma virus (RSV), suspended in vitreous ice, using transmission electron microscopy. The enveloped virions are spherical in shape, have a mean diameter of 127 nm, and vary significantly in size. Image processing reveals the presence of the viral matrix protein underlying the lipid bilayer and the viral envelope proteins external to the lipid bilayer. In the interior of the virus, the characteristic mature retroviral core is clearly imaged. In contrast to lentiviruses, such as human immunodeficiency virus, the core of RSV is essentially isometric. The capsid, or external shell of the core, has a faceted, almost polygonal appearance in electron micrographs, but many capsids also exhibit continuous surface curvature. Cores are not uniform in size or shape. Serrations observed along the projected faces of the core suggest a repetitive molecular structure. Some isolated cores were observed in the sample, confirming that cores are at least transiently stable in the absence of the viral envelope. Using an approach grounded in geometric probability, we estimate the size of the viral core from the projection data. We show that the size of the core is not tightly controlled and that core size and virion size are positively correlated. From estimates of RNA packing density we conclude that either the RNA within the core is loosely packed or, more probably, that it does not fill the core.
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Human bile salt-stimulated lipase (BSSL), which is secreted from the pancreas into the digestive tract and from the lactating mammary gland into human milk, is important for the effective absorption of dietary lipids. The dependence of... more
Human bile salt-stimulated lipase (BSSL), which is secreted from the pancreas into the digestive tract and from the lactating mammary gland into human milk, is important for the effective absorption of dietary lipids. The dependence of BSSL on bile acids for activity with water-insoluble substrates differentiates it from other lipases. We have determined the crystal structure of a truncated variant of human BSSL (residues 1-5.8) and refined it at 2.60 A resolution, to an R-factor of 0.238 and R(free) of 0.275. This variant lacks the C-terminal alpha-helix and tandem C-terminal repeat region of native BSSL, but retains full catalytic activity. A short loop (residues 115-126) capable of occluding the active-site (the active site loop) is highly mobile and exists in two conformations, the most predominant of which leaves the active-site open for interactions with substrate. The bile salt analogue 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid (CHAPS) was present in the crystallisation medium, but was not observed bound to the enzyme. However, the structure reveals a sulfonate group from the buffer piperizine ethane sulfonic acid (PIPES), making interactions with Arg63 and His115. His115 is part of the active-site loop, indicating that the loop could participate in the binding of a sulphate group from either the glycosaminoglycan heparin (known to bind BSSL) or a bile acid such as deoxycholate. Opening of the 115-126 active-site loop may be cooperatively linked to a sulphate anion binding at this site. The helix bundle domain of BSSL (residues 319-398) exhibits weak electron density and high temperature factors, indicating considerable structural mobility. This domain contains an unusual Asp:Glu pair buried in a hydrophobic pocket between helices alpha(H) and alpha(K) that may be functionally important. We have also solved the structure of full-length glycosylated human BSSL at 4.1 A resolution, using the refined coordinates of the truncated molecule as a search model. This structure reveals the position of the C-terminal helix, missing in the truncated variant, and also shows the active-site loop to be in a closed conformation.
Research Interests: Molecular Biology, Crystallization, Macromolecular X-Ray Crystallography, Molecular, Crystal structure, and 21 moreHumans, Heparin, Animals, High Temperature, Glycosaminoglycan, Electron Density, Enzyme, Glycosylation, Cattle, Digestive Tract, Active site, Mammary gland, Human Milk, Protein Conformation, Recombinant Proteins, Protein Binding, Catalytic Activity, Full Length Movies, Biochemistry and cell biology, Solvents, and Pliability(Humans, Heparin, Animals, High Temperature, Glycosaminoglycan, Electron Density, Enzyme, Glycosylation, Cattle, Digestive Tract, Active site, Mammary gland, Human Milk, Protein Conformation, Recombinant Proteins, Protein Binding, Catalytic Activity, Full Length Movies, Biochemistry and cell biology, Solvents, and Pliability)
(Humans, Heparin, Animals, High Temperature, Glycosaminoglycan, Electron Density, Enzyme, Glycosylation, Cattle, Digestive Tract, Active site, Mammary gland, Human Milk, Protein Conformation, Recombinant Proteins, Protein Binding, Catalytic Activity, Full Length Movies, Biochemistry and cell biology, Solvents, and Pliability)
Human bile salt-stimulated lipase (BSSL), which is secreted from the pancreas into the digestive tract and from the lactating mammary gland into human milk, is important for the effective absorption of dietary lipids. The dependence of... more
Human bile salt-stimulated lipase (BSSL), which is secreted from the pancreas into the digestive tract and from the lactating mammary gland into human milk, is important for the effective absorption of dietary lipids. The dependence of BSSL on bile acids for activity with water-insoluble substrates differentiates it from other lipases. We have determined the crystal structure of a truncated variant of human BSSL (residues 1-5.8) and refined it at 2.60 A resolution, to an R-factor of 0.238 and R(free) of 0.275. This variant lacks the C-terminal alpha-helix and tandem C-terminal repeat region of native BSSL, but retains full catalytic activity. A short loop (residues 115-126) capable of occluding the active-site (the active site loop) is highly mobile and exists in two conformations, the most predominant of which leaves the active-site open for interactions with substrate. The bile salt analogue 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid (CHAPS) was present in the crystallisation medium, but was not observed bound to the enzyme. However, the structure reveals a sulfonate group from the buffer piperizine ethane sulfonic acid (PIPES), making interactions with Arg63 and His115. His115 is part of the active-site loop, indicating that the loop could participate in the binding of a sulphate group from either the glycosaminoglycan heparin (known to bind BSSL) or a bile acid such as deoxycholate. Opening of the 115-126 active-site loop may be cooperatively linked to a sulphate anion binding at this site. The helix bundle domain of BSSL (residues 319-398) exhibits weak electron density and high temperature factors, indicating considerable structural mobility. This domain contains an unusual Asp:Glu pair buried in a hydrophobic pocket between helices alpha(H) and alpha(K) that may be functionally important. We have also solved the structure of full-length glycosylated human BSSL at 4.1 A resolution, using the refined coordinates of the truncated molecule as a search model. This structure reveals the position of the C-terminal helix, missing in the truncated variant, and also shows the active-site loop to be in a closed conformation.
Research Interests: Molecular Biology, Crystallization, Macromolecular X-Ray Crystallography, Molecular, Crystal structure, and 21 moreHumans, Heparin, Animals, High Temperature, Glycosaminoglycan, Electron Density, Enzyme, Glycosylation, Cattle, Digestive Tract, Active site, Mammary gland, Human Milk, Protein Conformation, Recombinant Proteins, Protein Binding, Catalytic Activity, Full Length Movies, Biochemistry and cell biology, Solvents, and Pliability(Humans, Heparin, Animals, High Temperature, Glycosaminoglycan, Electron Density, Enzyme, Glycosylation, Cattle, Digestive Tract, Active site, Mammary gland, Human Milk, Protein Conformation, Recombinant Proteins, Protein Binding, Catalytic Activity, Full Length Movies, Biochemistry and cell biology, Solvents, and Pliability)
(Humans, Heparin, Animals, High Temperature, Glycosaminoglycan, Electron Density, Enzyme, Glycosylation, Cattle, Digestive Tract, Active site, Mammary gland, Human Milk, Protein Conformation, Recombinant Proteins, Protein Binding, Catalytic Activity, Full Length Movies, Biochemistry and cell biology, Solvents, and Pliability)
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Research Interests: Electron Microscopy, Structure, Nuclear Magnetic Resonance, Self Assembly, NMR Spectroscopy, and 12 moreMagnetic Resonance Spectroscopy, Macromolecular X-Ray Crystallography, Biological Sciences, Humans, Capsid, CHEMICAL SCIENCES, Human immunodeficiency virus, Protein Secondary Structure Prediction, Similarity Solution, Long Range Order, X Ray Crystallography, and Capsid Protein
Research Interests: Molecular Biology, Structural Biology, Protein Folding, Virology, Crystallization, and 34 moreFungi, Cryopreservation, Transcription Factors, Macromolecular X-Ray Crystallography, Ruminants, Antirrhinum, Biological Sciences, DNA, Molecular chaperones, South Africa, Magnesium, Diffusion, Sheep, Animals, Rhodococcus, Pseudomonas, Physical sciences, Temperature, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Goats, CHEMICAL SCIENCES, Cattle, Molecular cloning, X ray diffraction, Protein Conformation, Protein Binding, Chitin, Klebsiella Oxytoca, Ethylene Glycol, Biochemistry and cell biology, Solvents, Adenosine Triphosphate, Adenosine Diphosphate, and Bluetongue virus(Fungi, Cryopreservation, Transcription Factors, Macromolecular X-Ray Crystallography, Ruminants, Antirrhinum, Biological Sciences, DNA, Molecular chaperones, South Africa, Magnesium, Diffusion, Sheep, Animals, Rhodococcus, Pseudomonas, Physical sciences, Temperature, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Goats, CHEMICAL SCIENCES, Cattle, Molecular cloning, X ray diffraction, Protein Conformation, Protein Binding, Chitin, Klebsiella Oxytoca, Ethylene Glycol, Biochemistry and cell biology, Solvents, Adenosine Triphosphate, Adenosine Diphosphate, and Bluetongue virus)
(Fungi, Cryopreservation, Transcription Factors, Macromolecular X-Ray Crystallography, Ruminants, Antirrhinum, Biological Sciences, DNA, Molecular chaperones, South Africa, Magnesium, Diffusion, Sheep, Animals, Rhodococcus, Pseudomonas, Physical sciences, Temperature, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Goats, CHEMICAL SCIENCES, Cattle, Molecular cloning, X ray diffraction, Protein Conformation, Protein Binding, Chitin, Klebsiella Oxytoca, Ethylene Glycol, Biochemistry and cell biology, Solvents, Adenosine Triphosphate, Adenosine Diphosphate, and Bluetongue virus)
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Research Interests: Structure, Molecular Evolution, Macromolecular X-Ray Crystallography, Biological Sciences, Crystal structure, and 15 moreFace to Face, Oxidoreductases, Ethanol Production, Osmotic pressure, Enzyme, CHEMICAL SCIENCES, Hydrogen Bonding, Active site, Protein Secondary Structure Prediction, Protein Conformation, Amino Acid Sequence, Structural Similarity Index, X Ray Crystallography, Zymomonas, and Molecular Sequence Data(Face to Face, Oxidoreductases, Ethanol Production, Osmotic pressure, Enzyme, CHEMICAL SCIENCES, Hydrogen Bonding, Active site, Protein Secondary Structure Prediction, Protein Conformation, Amino Acid Sequence, Structural Similarity Index, X Ray Crystallography, Zymomonas, and Molecular Sequence Data)
(Face to Face, Oxidoreductases, Ethanol Production, Osmotic pressure, Enzyme, CHEMICAL SCIENCES, Hydrogen Bonding, Active site, Protein Secondary Structure Prediction, Protein Conformation, Amino Acid Sequence, Structural Similarity Index, X Ray Crystallography, Zymomonas, and Molecular Sequence Data)
In protein crystallography, the initial experimental problem is the identification of physical and chemical conditions that will support nucleation and crystal growth. Ideally, experiments to search for such conditions would be based on a... more
In protein crystallography, the initial experimental problem is the identification of physical and chemical conditions that will support nucleation and crystal growth. Ideally, experiments to search for such conditions would be based on a full-factorial structure, with variation in the temperature and solution composition. However, consideration of even a moderate number of possibilities for the composition of the system will result in factorial experiments which may be prohibitively large. In this paper it is proposed that search experiments for protein crystallization might be based on orthogonal arrays. These are subsets of full-factorial experiments which possess a great deal of symmetry, such that a uniform distribution of points throughout the experimental region is preserved. Such experiments have reasonable size, explore the proposed experimental region in a systematic fashion, and form a logical basis for a sequential approach to the search for crystallization conditions. Examples of such initial search experiments are given, and their application to some recent protein crystallization problems in this laboratory is described briefly. The relationship of this approach to other protein crystallization search procedures is also discussed.