Structure of cubic insulin crystals in glucose solutions.

X-ray structures of cubic insulin crystals in high concentrations of glucose at different pH levels and temperatures have been refined to high resolution. We have identified one glucose-binding site near the N-terminus of the A-chain whose occupancy is pH dependent. The effects of reduced water activity on the ordered protein and solvent structures have been examined. Our analysis showed no notable conformational changes in the ordered protein structures or ordered solvent molecules near the protein surface, but the presence of glucose does have a significant effect on the overall density distribution of the bulk solvent in the solvent-accessible volume. We compared the structure of cubic insulin at room temperature and liquid-nitrogen temperature, under identical solvent conditions, using glucose as a cryoprotectant. In this case, we found that the average temperature factor of the protein is reduced and more water molecules can be identified, but there are no significant changes in the protein conformation.     

High pressure macromolecular crystallography: the 140-MPa crystal structure at 2.3 A resolution of urate oxidase, a 135-kDa tetrameric assembly

We report the three-dimensional structure determined by high-pressure macromolecular crystallography (HPMX) of a 135-kDa homo-tetrameric enzyme, urate oxidase from Aspergillus flavus complexed with its potent inhibitor 8-azaxanthin. Urate oxidase crystals are quite sensitive to pressure, as three-dimensional order is lost at about 180 MPa. A highly complete 2.3 A resolution data set was collected at 140 MPa, close to the critical pressure. Crystal structures at atmospheric pressure and at high pressure were refined in the orthorhombic space group I222 with final crystallographic R factors 14.1% and 16.1%, respectively. The effect of pressure on temperature factors, ordered water molecules, hydrogen bond lengths, contacts, buried surface areas as well as cavity volume was investigated. Results suggest that the onset of disruption of the tetrameric assembly by pressure has been captured in the crystalline state.     

Structural changes within unit cell subunits in crosslinked alpha-chymotrypsin crystals.

The structural effects of bifunctional and polyfunctional protein crosslinking agents on protein molecules are, in principle, studied most exactly by modifying and examining a repeating ordered array of protein molecules, i.e. as in single crystals. While X-ray crystallography was possible with crosslinked carboxypeptidase A (Quiocho &; Richards, 1964), with other protein crystals disorder frequently obscured any reasonably resolvable electron density map. We have examined α-chymotrypsin crystals using electron spin resonance spin labels as a probe of the active site region only, i.e. observing one local region of a protein (crystal) structure in the midst of a (potentially) overall disordered structure. The methods and techniques of handling spin-labeled protein crystals were outlined by Bauer &; Berliner (1979).     

Evolutionary rate heterogeneity in proteins with long disordered regions

The dominant view in protein science is that a three-dimensional (3-D) structure is a prerequisite for protein function. In contrast to this dominant view, there are many counterexample proteins that fail to fold into a 3-D structure, or that have local regions that fail to fold, and yet carry out function. Protein without fixed 3-D structure is called intrinsically disordered. Motivated by anecdotal accounts of higher rates of sequence evolution in disordered protein than in ordered protein we are exploring the molecular evolution of disordered proteins. To test whether disordered protein evolves more rapidly than ordered protein, pairwise genetic distances were compared between the ordered and the disordered regions of 26 protein families having at least one member with a structurally characterized region of disorder of 30 or more consecutive residues. For five families, there were no significant differences in pairwise genetic distances between ordered and disordered sequences. The disordered region evolved significantly more rapidly than the ordered region for 19 of the 26 families. The functions of these disordered regions are diverse, including binding sites for protein, DNA, or RNA and also including flexible linkers. The functions of some of these regions are unknown. The disordered regions evolved significantly more slowly than the ordered regions for the two remaining families. The functions of these more slowly evolving disordered regions include sites for DNA binding. More work is needed to understand the underlying causes of the variability in the evolutionary rates of intrinsically ordered and disordered protein.     

The Use of Electron Tomography for Structural Analysis of Disordered Protein Arrays

A method based on Fourier transforms is described for obtaining a 3-D reconstruction from a paracrystalline object with static disorder. The method is derived from the standard methods used in 3-D reconstruction of 2-D crystals except that all of the Fourier coefficients are used and not just the sampled data from the periodic lattice. Thus, not only is the spatially ordered part of the structure visualized in 3-D, but also the spatially disordered part. Application of the method to 3-D reconstructions of insect flight muscle is described as well as prospects for extension of the method to radiation-sensitive specimens.     

Structure and order of the protein and carbohydrate domains of prothrombin fragment 1

The three-dimensional structure of prothrombin fragment 1 has been determined by X-ray crystallography at 3.8 A resolution. The fragment is composed of a number of structural units, some of which are ordered while others are disordered. The ordered part of the structure includes a compact kringle unit, a helical domain and a carbohydrate chain. The kringle structure is organized around a close pair of buried disulfide bridges. One of its carbohydrate chains, that attached to Asn 101, is fully ordered, but the carbohydrate chain attached to Asn 77 appears to be disordered. The calcium binding unit is composed of a disordered part containing all ten gamma-carboxyglutamic acid residues and an ordered part forming the helical domain. The highly conserved residues Phe 41, Trp 42 and Tyr 45, which form a hydrophobic cluster on the first helix, interact around a crystallographic two-fold axis with the equivalent residues in another molecule to form a dimer in the crystal.     

Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts

Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight.     

Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.     

Opening the high-pressure domain beyond 2 kbar to protein and virus crystallography--technical advance

The combined use of a diamond anvil cell and ultrashort-wavelength undulator radiation has allowed the collection of high-resolution diffraction data from protein and virus crystals submitted to hydrostatic pressures beyond 2 kbar. Crystals of cubic cowpea mosaic virus (CPMV) can be compressed to at least 3.5 kbar. Diffraction from CPMV crystals displaying an unusual disorder at atmospheric pressure was considerably enhanced by application of pressure. These experiments suggest that pressure may be used in some cases to improve order in crystals.     

Comparison of lipid/gramicidin dispersions and cocrystals by Raman scattering

Gramicidin crystals, dimyristoylphosphatidylcholine (DMPC)/gramicidin dispersions, and DMPC/gramicidin cocrystals were examined by Raman scattering to determine lipid/gramicidin stoichiometries and lipid organization. Calibrations of the choline (716-cm-1) and tryptophan (756-cm-1) peaks indicate that the cocrystals contain two lipids for each gramicidin monomer, a result confirmed by chemical analyses of washed crystals. In dispersions with high lipid/gramicidin ratios (e.g., 25:1), the lipid is ordered but becomes increasingly disordered as the gramicidin content is increased. Paradoxically, the DMPC/gramicidin cocrystals have highly ordered lipids that possibly contain no gauche bonds at all, despite their low lipid/gramicidin ratio. In addition, the polypeptide amide I peak position near 1670 cm-1 is found to be independent of the lipid/gramicidin ratio in the complexes and may indicate a beta-helix-type secondary structure at all ratios. However, the amide I peak broadens significantly at low lipid/gramicidin ratios and broadens still further in the cocrystals, suggesting that protein-protein interactions may induce band-broadening distortions of the polypeptide structure.     

Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.     

Is there a biological cost of protein disorder? Analysis of cancer-associated mutations

As many diseases can be traced back to altered protein function, studying the effect of genetic variations at the level of proteins can provide a clue to understand how changes at the DNA level lead to various diseases. Cellular processes rely not only on proteins with well-defined structure but can also involve intrinsically disordered proteins (IDPs) that exist as highly flexible ensembles of conformations. Disordered proteins are mostly involved in signaling and regulatory processes, and their functional repertoire largely complements that of globular proteins. However, it was also suggested that protein disorder entails an increased biological cost. This notion was supported by a set of individual IDPs involved in various diseases, especially in cancer, and the increased amount of disorder observed among disease-associated proteins. In this work, we tested if there is any biological risk associated with protein disorder at the level of single nucleotide mutations. Specifically, we analyzed the distribution of mutations within ordered and disordered segments. Our results demonstrated that while neutral polymorphisms were more likely to occur within disordered segments, cancer-associated mutations had a preference for ordered regions. Additionally, we proposed an alternative explanation for the association of protein disorder and the involvement in cancer with the consideration of functional annotations. Individual examples also suggested that although disordered segments are fundamental functional elements, their presence is not necessarily accompanied with an increased mutation rate in cancer. The presented study can help to understand how the different structural properties of proteins influence the consequences of genetic mutations.     

Temperature and ligand dependence of conformation and helical order in myosin filaments

Mammalian myosin filaments are helically ordered only at higher temperatures (>20 degrees C) and become progressively more disordered as the temperature is decreased. It had previously been suggested that this was a consequence of the dependence of the hydrolytic step of myosin ATPase on temperature and the requirement that hydrolysis products (e.g., ADP.P(i)) be bound at the active site. An alternative hypothesis is that temperature directly affects the conformation of the myosin heads and that they need to be in a particular conformation for helical order in the filament. To discriminate between these two hypotheses, we have studied the effect of temperature on the helical order of myosin heads in rabbit psoas muscle in the presence of nonhydrolyzable ligands. The muscle fibers were overstretched to nonoverlap such that myosin affinity for nucleotides was not influenced by the interaction of myosin with the thin filament. We show that with bound ADP.vanadate, which mimics the transition state between ATP and hydrolysis products, or with the ATP analogues AMP-PNP or ADP.BeF(x)() the myosin filaments are substantially ordered at higher temperatures but are reversibly disordered by cooling. These results reinforce recent studies in solution showing that temperature as well as ligand influence the equilibrium between multiple myosin conformations [Málnási-Csizmadia, A., Pearson, D. S., Kovács, M., Woolley, R. J., Geeves, M. A., and Bagshaw, C. R. (2001) Biochemistry 40, 12727-12737; Málnási-Csizmadia, A., Woolley, R. J., and Bagshaw, C. R. (2000) Biochemistry 39, 16135-16146; Urbanke, C., and Wray, J. (2001) Biochem. J. 358, 165-173] and indicate that helical order requires the myosin heads to be in the closed conformation. Our results suggest that most of the heads in the closed conformation are ordered, and that order is not produced in a separate step. Hence, helical order can be used as a signature of the closed conformation in relaxed muscle. Analysis of the dependence on temperature of helical order and myosin conformation shows that in the presence of these analogues one ordered (closed) conformation and two disordered conformations with distinct thermodynamic properties coexist. Low temperatures favor one disordered conformation, while high temperatures favor the ordered (closed) conformation together with a second disordered conformation.     

TOP-IDP-scale: a new amino acid scale measuring propensity for intrinsic disorder

Intrinsically disordered proteins carry out various biological functions while lacking ordered secondary and/or tertiary structure. In order to find general intrinsic properties of amino acid residues that are responsible for the absence of ordered structure in intrinsically disordered proteins we surveyed 517 amino acid scales. Each of these scales was taken as an independent attribute for the subsequent analysis. For a given attribute value X, which is averaged over a consecutive string of amino acids, and for a given data set having both ordered and disordered segments, the conditional probabilities P(s(o) | x) and P(s(d) | x) for order and disorder, respectively, can be determined for all possible values of X. Plots of the conditional probabilities P(s(o) | x) and P(s(o) | x) versus X give a pair of curves. The area between these two curves divided by the total area of the graph gives the area ratio value (ARV), which is proportional to the degree of separation of the two probability curves and, therefore, provides a measure of the given attribute's power to discriminate between order and disorder. As ARV falls between zero and one, larger ARV corresponds to the better discrimination between order and disorder. Starting from the scale with the highest ARV, we applied a simulated annealing procedure to search for alternative scale values and have managed to increase the ARV by more than 10%. The ranking of the amino acids in this new TOP-IDP scale is as follows (from order promoting to disorder promoting): W, F, Y, I, M, L, V, N, C, T, A, G, R, D, H, Q, K, S, E, P. A web-based server has been created to apply the TOP-IDP scale to predict intrinsically disordered proteins (http://www.disprot.org/dev/disindex.php).     

Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump

Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with phospholamban. In earlier studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of phospholamban and the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps from negatively stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of phospholamban was correlated with an order-to-disorder transition of the phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 Å, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association.     

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of ∼3.5 Å in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.     

A preformed, ordered structure of a 25-residue peptide derived from a major histocompatibility complex class I antigen is required to affect insulin receptor function

It was recently shown that a 25-residue peptide, Dk-(61-85), derived from the alpha 1 domain of a murine major histocompatibility class I molecule (H-2Dk), affects insulin receptor functions (Hansen, T., Stagsted, J., Pedersen, L., Roth, R. A., Goldstein, A., and Olsson, L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3123-3126; Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now report that this peptide can reversibly assume a biologically active or inactive state as measured in the rat adipocyte glucose uptake assay, implying that the peptide has at least two interconvertible conformations. The peptide has an ordered conformation in 0.1 M HCl or 0.1 M NaCl stock solution as shown by circular dichroism, but has a disordered molecular structure and is inactive when dissolved in H2O. The biologically active peptide forms liquid crystals at the stock solution concentration (1 mM), so the CD spectra do not provide information on the secondary structure. Under all conditions tested, biological activity (measured after transfer to assay buffer) is associated with an ordered conformation in stock solution. Biological activity and an ordered conformation of the peptide in H2O stock solution can be induced by increasing ionic strength (greater than 100 mM NaCl for maximal effect) or increasing pH (greater than 5 for maximal effect). The induction rate of the ordered conformation is slow with a half-maximal value obtained after approximately 20 min. Both biological activity and the ordered structure are lost upon heating of stock solution to 90 degrees C or upon transfer to assay buffer. A similar correlation of ordered structure with biological activity was observed with two truncated peptides derived from Dk-(61-85). It is inferred from these results that the Dk-(61-85) peptide and related peptides only affect insulin-stimulated glucose uptake in rat adipocytes if they have assumed an ordered conformation in stock solution prior to transfer to assay buffer and exposure to cells.     

水在稳定肌红蛋白天然结构中的作用

在０－１００％相对湿度（ＲＨ）范围内用付里叶变换红外光谱和曲线拟合法研究了水合对肌红蛋白二级结构的影响。干燥蛋白的α－螺旋、伸展和无序结构含量分别为６１％、２２％和１７％。随水合度增加,无序结构含量减少,有序二级结构含量增加,在４４％ＲＨ以下这种变化最显著,在８６－１００％ＲＨ范围,结构有较复杂的调整,三种亚结构含量分别约为７５％、２１％和４％,与晶态或溶液态下的结构相同。这一结果进一步证实了我们     

Protein flexibility and intrinsic disorder

Comparisons were made among four categories of protein flexibility: (1) low-B-factor ordered regions, (2) high-B-factor ordered regions, (3) short disordered regions, and (4) long disordered regions. Amino acid compositions of the four categories were found to be significantly different from each other, with high-B-factor ordered and short disordered regions being the most similar pair. The high-B-factor (flexible) ordered regions are characterized by a higher average flexibility index, higher average hydrophilicity, higher average absolute net charge, and higher total charge than disordered regions. The low-B-factor regions are significantly enriched in hydrophobic residues and depleted in the total number of charged residues compared to the other three categories. We examined the predictability of the high-B-factor regions and developed a predictor that discriminates between regions of low and high B-factors. This predictor achieved an accuracy of 70% and a correlation of 0.43 with experimental data, outperforming the 64% accuracy and 0.32 correlation of predictors based solely on flexibility indices. To further clarify the differences between short disordered regions and ordered regions, a predictor of short disordered regions was developed. Its relatively high accuracy of 81% indicates considerable differences between ordered and disordered regions. The distinctive amino acid biases of high-B-factor ordered regions, short disordered regions, and long disordered regions indicate that the sequence determinants for these flexibility categories differ from one another, whereas the significantly-greater-than-chance predictability of these categories from sequence suggest that flexible ordered regions, short disorder, and long disorder are, to a significant degree, encoded at the primary structure level.     

Effects of cholesterol on the structure and collapse of DPPC monolayers

Cholesterol induces faster collapse by compressed films of pulmonary surfactant. Because collapse prevents films from reaching the high surface pressures achieved in the alveolus, most therapeutic surfactants remove or omit cholesterol. The studies here determined the structural changes by which cholesterol causes faster collapse by films of dipalmitoyl phosphatidylcholine, used as a simple model for the functional alveolar film. Measurements of isobaric collapse, with surface pressure held constant at 52 mN/m, showed that cholesterol had little effect until the mol fraction of cholesterol, X_chol, exceeded 0.20. Structural measurements of grazing incidence X-ray diffraction at ambient laboratory temperatures and a surface pressure of 44 mN/m, just below the onset of collapse, showed that the major structural change in an ordered phase occurred at lower X_chol. A centered rectangular unit cell with tilted chains converted to an untilted hexagonal structure over the range of X_chol = 0.0–0.1. For X_chol = 0.1–0.4, the ordered structure was nearly invariant; the hexagonal unit cell persisted, and the spacing of the chains was essentially unchanged. That invariance strongly suggests that above X_chol = 0.1, cholesterol partitions into a disordered phase, which coexists with the ordered domains. The phase rule requires that for a binary film with coexisting phases, the stoichiometries of the ordered and disordered regions must remain constant. Added cholesterol must increase the area of the disordered phase at the expense of the ordered regions. X-ray scattering from dipalmitoyl phosphatidylcholine/cholesterol fit with that prediction. The data also show a progressive decrease in the size of crystalline domains. Our results suggest that cholesterol promotes adsorption not by altering the unit cell of the ordered phase but by decreasing both its total area and the size of individual crystallites.