Heat-induced unfolding of neocarzinostatin, a small all-beta protein investigated by small-angle X-ray scattering

Neocarzinostatin is an all-beta protein, 113 amino acid residues long, with an immunoglobulin-like fold. Its thermal unfolding has been studied by small-angle X-ray scattering. Preliminary differential scanning calorimetry and fluorescence measurements suggest that the transition is not a simple, two-state transition. The apparent radius of gyration is determined using three different approaches, the validity of which is critically assessed using our experimental data as well as a simple, two-state model. Similarly, each step of data analysis is evaluated and the underlying assumptions plainly stated. The existence of at least one intermediate state is formally demonstrated by a singular value decomposition of the set of scattering patterns. We assume that the pattern of the solution before the onset of the transition is that of the native protein, and that of the solution at the highest temperature is that of the completely unfolded protein. Given these, actually not very restrictive, boundary constraints, a least-squares procedure yields a scattering pattern of the intermediate state. However, this solution is not unique: a whole class of possible solutions is derived by adding to the previous linear combination of the native and completely unfolded states. Varying the initial conditions of the least-squares calculation leads to very similar solutions. Whatever member of the class is considered, the conformation of this intermediate state appears to be weakly structured, probably less than the transition state should be according to some proposals. Finally, we tried and used the classical model of three thermodynamically well-defined states to account for our data. The failure of the simple thermodynamic model suggests that there is more than the single intermediate structure required by singular value decomposition analysis. Formally, there could be several discrete intermediate species at equilibrium, or an ensemble of conformations differently populated according to the temperature. In the latter case, a third state would be a weighted average of all non native and not completely unfolded states of the protein but, since the weights change with temperature, no meaningful curve is likely to be derived by a global analysis using the simple model of three thermodynamically well-defined states.     

Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.     

Solution structural characterization of coiled-coil peptide-polymer side-conjugates

Detailed structural characterization of protein-polymer conjugates and understanding of the interactions between covalently attached polymers and biomolecules will build a foundation to design and synthesize hybrid biomaterials. Conjugates based on simple protein structures are ideal model system to achieve these ends. Here we present a systematic structural study of coiled-coil peptide-poly(ethylene glycol) (PEG) side-conjugates in solution, using circular dichroism, dynamic light scattering, and small-angle X-ray scattering, to determine the conformation of conjugated PEG chains. The overall size and shape of side-conjugates were determined using a cylindrical form factor model. Detailed structural information of the covalently attached PEG chains was extracted using a newly developed model where each peptide-PEG conjugate was modeled as a Gaussian chain attached to a cylinder, which was further arranged in a bundle-like configuration of three or four cylinders. The peptide-polymer side-conjugates were found to retain helix bundle structure, with the polymers slightly compressed in comparison with the conformation of free polymers in solution. Such detailed structural characterization of the peptide-polymer conjugates, which elucidates the conformation of conjugated PEG around the peptide and assesses the effect of PEG on peptide structure, will contribute to the rational design of this new family of soft materials.     

Ligand-induced domain rearrangement of fatty acid beta-oxidation multienzyme complex

The quaternary structure of a fatty acid beta-oxidation multienzyme complex, catalyzing three sequential reactions, was investigated by X-ray crystallographic and small-angle X-ray solution scattering analyses. X-ray crystallography revealed an intermediate structure of the complex among the previously reported structures. However, the theoretical scattering curves calculated from the crystal structures remarkably disagree with the experimental profiles. Instead, an ensemble of the atomic models, which were all calculated by rigid-body optimization, reasonably explained the experimental data. These structures significantly differ from those in the crystals, but they maintain the substrate binding pocket at the domain boundary. Comparisons among these structures indicated that binding of 3-hydroxyhexadecanoyl-CoA or nicotinamide adenine dinucleotide induces domain rearrangements in the complex. The conformational changes suggest the structural events occurring during the chain reaction catalyzed by the multienzyme complex.     

Divalent ion-dependent swelling of tomato bushy stunt virus: a multi-approach study

Time-resolved small-angle X-ray and neutron scattering (SAXS and SANS) in solution were used to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. SAXS intensities comprise contribution from the protein capsid and the RNA moiety, while neutron scattering, recorded in 72% D2O, is essentially due to the protein capsid. Cryo-electron micrographs of compact and swollen virus were used to produce 3D reconstructions of the initial and final conformations of the virus at a resolution of 13 A and 19 A, respectively. While compact particles appear to be very homogeneous in size, solutions of swollen particles exhibit some size heterogeneity. A procedure has been developed to compute the SAXS pattern from the 3D reconstruction for comparison with experimental data. Cryo-electron microscopy thereby provides an invaluable starting (and ending) point for the analysis of the time-resolved swelling process using the scattering data.     

The Global Conformation of an Active Hammerhead RNA during the Process of Self-cleavage

The RNA “hammerhead” domain is a small element of secondary structure found in the genomes of certain plant pathogens. It possesses a core of conserved sequence at the conjunction of three helix stems, and is capable of undergoing self-cleavage in the presence of divalent cations. Both crystallographic and solution studies suggest that the domain is highly structured, with the three stems assuming a Y-shaped global conformation; however, such studies have employed either RNA analogues that were catalytically inactive, or conditions of temperature and pH for which rates of self-cleavage are slow. Thus, it was unknown whether such species represented the principal conformers during the cleavage process itself. In order to address this issue, a series of time- resolved, transient electric birefringence measurements was conducted in an effort to define the global conformation of an RNA hammerhead in real time throughout the process of self-cleavage. The current study demonstrates that the angular relationship between the two helices that flank the cleavage center is essentially unchanged between the pre-cleavage and post-cleavage forms. Moreover, despite the fact that at least one kinetic intermediate is formed during the self-cleavage reaction, there is no evidence for the existence of a significant population of intermediates with altered global conformation during cleavage. Thus, any conformational isomerism that may occur is likely to be relatively localized to the active center. Finally, it was observed that sequence elements lying outside of the conserved region, at the base of stem I, influence interhelix geometry. The current results are consistent with a structural model in which the active center possesses similar conformations pre-cleavage and post-cleavage. Such a model would help to explain the significant rate of reversal of the cleavage reaction (self-ligation).     

X-ray scattering combined with coordinate-based analyses for applications in natural and artificial photosynthesis

Advances in X-ray light sources and detectors have created opportunities for advancing our understanding of structure and structural dynamics for supramolecular assemblies in solution by combining X-ray scattering measurement with coordinate-based modeling methods. In this review the foundations for X-ray scattering are discussed and illustrated with selected examples demonstrating the ability to correlate solution X-ray scattering measurements to molecular structure, conformation, and dynamics. These approaches are anticipated to have a broad range of applications in natural and artificial photosynthesis by offering possibilities for structure resolution for dynamic supramolecular assemblies in solution that can not be fully addressed with crystallographic techniques, and for resolving fundamental mechanisms for solar energy conversion by mapping out structure in light-excited reaction states.     

Structural characterization of the active and inactive states of Src kinase in solution by small-angle X-ray scattering

Src kinase plays an important role in several signaling and regulation mechanisms in vivo. Enzymatic activity is tightly regulated through the phosphorylation and dephosphorylation of tyrosine 527, which is placed at the C-terminal tail. Here, we have addressed domain rearrangements involved in the regulation mechanism of Src kinase in solution using small-angle X-ray scattering. In the phosphorylated wild-type form of Src kinase corresponding to the inactive state of the protein, a single conformation compatible with a closed crystallographic structure was found in solution. In the Y527F point mutant representing the active state, analysis of scattering data reveals an equilibrium between two differently populated conformations differing in the radius of gyration by 5 Å. The major species (85% of the total population) presents a closed conformation indistinguishable from the crystallographic structure of the inactive state. The minor species (15% of the total population) is an open conformation similar to the crystallographic structure in the active state. The latter structure has the SH3, SH2, and SH2-catalytic domain linker assembled as a pseudo-two-domain protein. The regulation model emerging from this study, including at least three different conformational states, allows the tight regulation of the enzyme without compromising fast response in the presence of natural targets.     

Unlocked Concanavalin A Forms Amyloid-like Fibrils from Coagulation of Long-lived “Crinkled” Intermediates

Understanding the early events during amyloid aggregation processes is crucial to single out the involved molecular mechanisms and for designing ad hoc strategies to prevent and reverse amyloidogenic disorders. Here, we show that, in conditions in which the protein is positively charged and its conformational flexibility is enhanced, Concanavalin A leads to fibril formation via a non-conventional aggregation pathway. Using a combination of light scattering, circular dichroism, small angle X-ray scattering, intrinsic (Tryptophan) and extrinsic (ANS) fluorescence and confocal and 2-photon fluorescence microscopy we characterize the aggregation process as a function of the temperature. We highlight a multi-step pathway with the formation of an on-pathway long-lived intermediate and a subsequent coagulation of such “crinkled” precursors into amyloid-like fibrils. The process results in a temperature-dependent aggregation-coagulation pathway, with the late phase of coagulation determined by the interplay between hydrophobic and electrostatic forces. Our data provide evidence for the complex aggregation pathway for a protein with a highly flexible native conformation. We demonstrate the possibility to generate a long-lived intermediate whose proportion and occurrence are easily tunable by experimental parameters (i.e. temperature). As a consequence, in the case of aggregation processes developing through well-defined energy barriers, our results can open the way to new strategies to induce more stable in vitro on-pathway intermediate species through a minute change in the initial conformational flexibility of the protein. This will allow isolating and experimentally studying such transient species, often indicated as relevant in neurodegenerative diseases, both in terms of structural and cytotoxic properties.     

Modelling the surface lattice of alpha-keratin filaments

A recently-proposed model for the distribution of scattering material on the surface lattice of alpha-keratin intermediate filaments in dry porcupine quill is examined in detail. It is shown that, while retaining the basic form of the model (namely a dislocated helix with finite lattice spacing of 198.2 A), alternative meridonal distributions of scattering material within the finite lattice unit cell can be obtained which are consistent with the low-angle meridional X-ray pattern. The Gaussian shape function used to demonstrate the finite lattice in the model is questioned. The meridional diffraction pattern from hydrated porcupine quill is also examined and, apart from the intense fifth order reflection, can be modelled by distortion of the dry species scattering material distribution.     

Probing lysozyme conformation with light reveals a new folding intermediate

A means to control lysozyme conformation with light illumination has been developed using the interaction of the protein with a photoresponsive surfactant. Upon exposure to the appropriate wavelength of light, the azobenzene surfactant undergoes a reversible photoisomerization, with the visible-light (trans) form being more hydrophobic than the UV-light (cis) form. As a result, surfactant binding to the protein and, thus, protein unfolding, can be tuned with light. Small-angle neutron scattering (SANS) measurements were used to provide detailed information of the protein conformation in solution. Shape-reconstruction methods applied to the SANS data indicate that under visible light the protein exhibits a native-like form at low surfactant concentrations, a partially swollen form at intermediate concentrations, and a swollen/unfolded form at higher surfactant concentrations. Furthermore, the SANS data combined with FT-IR spectroscopic analysis of the protein secondary structure reveal that unfolding occurs primarily in the alpha domain of lysozyme, while the beta domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known alpha-domain intermediate of lysozyme that contains a folded alpha domain and unfolded beta domain. Because the interactions between the photosurfactant and protein can be tuned with light, illumination with UV light returns the protein to a native-like conformation. Fluorescence emission data of the nonpolar probe Nile red indicate that hydrophobic domains become available for probe partitioning in surfactant-protein solutions under visible light, while the availability of these hydrophobic domains to the probe decrease under UV light. Dynamic light scattering and UV-vis spectroscopic measurements further confirm the shape-reconstruction findings and reveal three discrete conformations of lysozyme. The results clearly demonstrate that visible light causes a greater degree of lysozyme swelling than UV light, thus allowing for the protein conformation to be controlled with light.     

Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering

Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.     

Structural dynamics in F1ATPase during the first reaction cycle of ATP hydrolysis

The velocity of ATP hydrolysis, catalyzed by purified F1ATPase from Micrococcus luteus, was decelerated on decreasing the temperature. At 13 degrees C one reaction cycle is completed after 20 s. Hydrolysis was triggered upon rapid mixing of the enzyme with ATP. During the first reaction cycle, succeeding structural alterations of the F1ATPase were traced by time resolved X-ray scattering. The scattering spectra obtained from consecutive intervals of 1 s, revealed the F1ATPase to pass a conformational state exhibiting an expanded (6%) molecular shape. The expanded state was observed between 45% and 65% of the time required to complete the reaction cycle. This points out a conformational pulsation during ATP hydrolysis.     

Oligomeric States along the Folding Pathways of β2-Microglobulin: Kinetics,Thermodynamics, and Structure

The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.     

Streptokinase is a flexible multi-domain protein

The structure of streptokinase in solution has been studied by dynamic light scattering, small-angle X-ray scattering and circular dichroism spectroscopy. The Stokes' radius and radius of gyration of the protein monomer are 3.58 nm and 4.03 nm, respectively. The maximum intraparticle distance of the molecule is 14 nm. More than half of the amino acids of the molecule are organized in regular secondary structures. The X-ray scattering curve, the results from dynamic light scattering, and the finding that at least 50% of the amino acid residues are organized in regularly folded secondary structures are consistent with the following structural model. Streptokinase consists of four compact, separately folded, domains linked by mobile segments of the protein chain. The molecule exhibits the conformation of a flexible string-of-beads in solution.     

Protein conformations explored by difference high-angle solution X-ray scattering: oxidation state and temperature dependent changes in cytochrome C

We demonstrate the use of high-angle X-ray scattering to explore protein conformational states in solution by resolving oxidation state- and temperature-dependent changes in the conformation of horse heart cytochrome c. Several detailed models exist for oxidation-dependent changes in mitochondrial class I c cytochromes determined by X-ray crystallography and solution NMR techniques. These models differ in the magnitude and locations of structural change. Our scattering measurements show that high-angle X-ray scattering can discriminate between these models, and that the experimental scattering data for horse cytochrome c can be best reconciled with selected NMR models for the same protein. These results demonstrate the ability to use high-angle X-ray scattering to resolve conformational states of proteins in solution, and to relate these measurements to detailed structural models. Furthermore, temperature-dependent changes are found in the high angle scattering patterns for horse cytochrome c, illustrating the sensitivity of these measurements to dynamic aspects of protein structure. These results demonstrate the ability to use difference high angle scattering as a quantitative monitor of reaction-linked changes in protein conformation and structural dynamics. Synchrotron-based high-angle scattering holds promise as a widely applicable, high throughput technique for exploring conformational states linked to physiological protein function, for resolving configurational differences between protein structures in solution and crystalline states, and for bridging the gap between solution NMR and crystallographic structure techniques.     

Small-angle X-ray scattering study of the (Fab′)2 fragment of the human immunoglobulin Kol

The conformation of the (Fab′)₂ fragment of the human immunoglubulin Kol has been investigated in solution by small angle X-ray scattering. The following molecular parameters were determined: radius of gyration 4.10 ± 0.05 nm; maximum distance 14.0 ± 0.5 nm and hydrated volume 150 ± 8 nm³. A model of the fragment is presented, which fits these experimental data and shows good agreement with the distance distribution function in real space and the scattering curve in reciprocal space. We have to assume that the (Fab′)₂ fragment has many different conformations in solution. The method of small-angle X-ray scattering only allows the determination of an average conformation which is very similar within the resolution of the method to the static structure determined in the crystal.     

Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering,Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation

The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution.     

Direct observation of three conformations of MutS protein regulated by adenine nucleotides

Mismatched base-pairs, which are caused by either DNA replication errors, DNA damage or genetic recombination, are repaired by the mismatch-repair system. The MutS protein, a component of the mismatch-repair system, recognizes mismatched base-pairs in DNA, and its DNA-binding activity is affected by ATP and ADP. Here, we show that the MutS protein from Thermus thermophilus HB8 can have three different conformations in solution, based on direct observations made by small-angle X-ray scattering. The conformation of MutS in solution is drastically influenced by the presence of ADP and ATP; the ATP-bound form has the most compact conformation, the ADP-bound form the most stretched, and the nucleotide-free form has a conformation intermediate between the two. Based on these findings, we conclude that the DNA-binding activity of MutS may depend on conformational changes triggered by both the binding and hydrolysis of ATP.     

Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes

Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 ? from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.