Single-shot NMR measurement of protein unfolding landscapes.

The transient unfolding events from the native state of a protein towards higher energy states can be closely investigated by studying the process of hydrogen exchange. Here, we present BLUU-Tramp (Biophysics Laboratory University of Udine-Temperature ramp), a new method to measure the rates for the exchange process and the underlying equilibrium thermodynamic parameters, using just a single sample preparation, in a single experiment that lasts some 20 to 60h depending on the protein thermal stability, to record hundreds of points over a virtually continuous temperature window. The method is suitable also in presence of other proteins in the sample, if only the target protein is (15)N-labelled. This allows the complete thermodynamic description of the unfolding landscape at an atomic level in the presence of small or macromolecular ligands or cosolutes, or in physiological environments. The method was successfully tested with human ubiquitin. Then the unfolding thermodynamic parameters were satisfactorily determined for the amyloidogenic protein ß(2)-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints.

Reduction of conformational mobility and aggregation in W60G ß2-microglobulin: assessment by 15N NMR relaxation.

The amyloid pathology associated with long-term haemodialysis is due to the deposition of ß2-microglobulin, the non-polymorphic light chain of class I major histocompatibility complex, that accumulates at bone joints into amyloid fibrils. Several lines of evidence show the relevance of the tryptophan residue at position 60 for the fibrillogenic transition of the protein. A comparative (15)N NMR relaxation analysis is presented for wild-type human ß2-microglobulin and W60G ß2-microglobulin, i.e. the mutant with a glycyne replacing the natural tryptophan residue at position 60. The experimental data, collected at 11.4 T and 310 K, were analyzed by means of the reduced spectral density approach. Molecular dynamics (MD) simulations and corresponding thermodynamic integration, together with hydrodynamic calculations were performed to support data interpretation. The analysis results for the mutant protein are consistent with a reduced aggregation with respect to the wild-type counterpart, as a consequence of an increased conformational rigidity probed by either NMR relaxation and MD simulations. Although dynamics in solution is other than fibrillar competence, the assessed properties of the mutant protein can be related with its reduced ability of forming fibrils when seeded in 20% trifluoroethanol.

Bluues: a program for the analysis of the electrostatic properties of proteins based on generalized Born radii.

BACKGROUND: The Poisson-Boltzmann (PB) equation and its linear approximation have been widely used to describe biomolecular electrostatics. Generalized Born (GB) models offer a convenient computational approximation for the more fundamental approach based on the Poisson-Boltzmann equation, and allows estimation of pairwise contributions to electrostatic effects in the molecular context. RESULTS: We have implemented in a single program most common analyses of the electrostatic properties of proteins. The program first computes generalized Born radii, via a surface integral and then it uses generalized Born radii (using a finite radius test particle) to perform electrostaic analyses. In particular the ouput of the program entails, depending on user's requirement: 1) the generalized Born radius of each atom; 2) the electrostatic solvation free energy; 3) the electrostatic forces on each atom (currently in a developmental stage); 4) the pH-dependent properties (total charge and pH-dependent free energy of folding in the pH range -2 to 18; 5) the pKa of all ionizable groups; 6) the electrostatic potential at the surface of the molecule; 7) the electrostatic potential in a volume surrounding the molecule; CONCLUSIONS: Although at the expense of limited flexibility the program provides most common analyses with requirement of a single input file in PQR format. The results obtained are comparable to those obtained using state-of-the-art Poisson-Boltzmann solvers. A Linux executable with example input and output files is provided as supplementary material.

Determining the energy landscape of proteins by a fast isotope exchange NMR approach.

We present a new and efficient NMR method, BLUU-Tramp (Biophysics Laboratory University of Udine temperature ramp), for the collection of hydrogen-deuterium exchange experiments as a function of time and temperature for small and medium-sized proteins. Exchange rates can be determined to extract the underlying thermodynamic equilibrium or kinetic parameters by sampling hundreds of points over a virtually continuous temperature ramp. Data are acquired in a single experimental session that lasts some 20-60 h, depending on the thermal stability of the protein. Subsequent analysis provides a complete thermodynamic description of the protein energy landscape. The global thermal unfolding process and the partial or local structure opening events can be fully determined at the single-residue resolution level. The proposed approach is shown to work successfully with the amyloidogenic protein ß(2)-microglobulin. With (15)N-labeling, the unfolding landscape of a protein can also be studied in the presence of other unlabeled proteins and, in general, with ligands or cosolutes or in physiological environments.

Studying interactions by molecular dynamics simulations at high concentration.

Molecular dynamics simulations have been used to study molecular encounters and recognition. In recent works, simulations using high concentration of interacting molecules have been performed. In this paper, we consider the practical problems for setting up the simulation and to analyse the results of the simulation. The simulation of beta 2-microglobulin association and the simulation of the binding of hydrogen peroxide by glutathione peroxidase are provided as examples.

Structural and dynamics characteristics of acylphosphatase from Sulfolobus solfataricus in the monomeric state and in the initial native-like aggregates.

It has previously been shown that the acylphosphatase from Sulfolobus solfataricus is capable of forming amyloid-like aggregates under conditions in which the native structure is maintained and via the transient formation of native-like aggregates. Based on the previously determined NMR structure of the native protein, showing a ferredoxin-like fold and the peculiar presence of an unstructured N-terminal segment, we show here, at a molecular level using NMR spectroscopy, that indeed S. solfataricus acylphosphatase remains in a native-like conformation when placed in aggregating conditions and that such a native-like structure persists when the protein forms the initial aggregates, at least within the low molecular weight species. The analysis carried out under different solution conditions, based on the measurement of the combined (1)H and (15)N chemical shifts and hydrogen/deuterium exchange rates, enabled the most significant conformational changes to be monitored upon transfer of the monomeric state into aggregating conditions and upon formation of the initial native-like aggregates. Important increases of the hydrogen/deuterium exchange rates throughout the native protein, accompanied by small and localized structural changes, in the monomeric protein were observed. The results also allow the identification of the intermolecular interaction regions within the native-like aggregates, that involve, in particular, the N-terminal unstructured segment, the apical region including strands S4 and S5 with the connecting loop, and the opposite active site.

Native-unlike long-lived intermediates along the folding pathway of the amyloidogenic protein beta2-microglobulin revealed by real-time two-dimensional NMR.

Beta2-microglobulin (beta2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified beta2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G beta2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G beta2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the beta2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type beta2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G beta2m mutant.

Molecular dynamics simulation of ß2-microglobulin in denaturing and stabilizing conditions.

ß2-Microglobulin has been a model system for the study of fibril formation for 20 years. The experimental study of ß2-microglobulin structure, dynamics, and thermodynamics in solution, at atomic detail, along the pathway leading to fibril formation is difficult because the onset of disorder and aggregation prevents signal resolution in Nuclear Magnetic Resonance experiments. Moreover, it is difficult to characterize conformers in exchange equilibrium. To gain insight (at atomic level) on processes for which experimental information is available at molecular or supramolecular level, molecular dynamics simulations have been widely used in the last decade. Here, we use molecular dynamics to address three key aspects of ß2-microglobulin, which are known to be relevant to amyloid formation: (1) 60 ns molecular dynamics simulations of ß2-microglobulin in trifluoroethanol and in conditions mimicking low pH are used to study the behavior of the protein in environmental conditions that are able to trigger amyloid formation; (2) adaptive biasing force molecular dynamics simulation is used to force cis-trans isomerization at Proline 32 and to calculate the relative free energy in the folded and unfolded state. The native-like trans-conformer (known as intermediate 2 and determining the slow phase of refolding), is simulated for 10 ns, detailing the possible link between cis-trans isomerization and conformational disorder; (3) molecular dynamics simulation of highly concentrated doxycycline (a molecule able to suppress fibril formation) in the presence of ß2-microglobulin provides details of the binding modes of the drug and a rationale for its effect.

Equilibrium unfolding thermodynamics of beta2-microglobulin analyzed through native-state H/D exchange.

The exchange rates for the amide hydrogens of beta(2)-microglobulin, the protein responsible for dialysis-related amyloidosis, were measured under native conditions at different temperatures ranging from 301 to 315 K. The pattern of protection factors within different regions of the protein correlates well with the hydrogen-bonding pattern of the deposited structures. Analysis of the exchange rates indicates the presence of mixed EX1- and EX2-limit mechanisms. The measured parameters are consistent with a two-process model in which two competing pathways, i.e., global unfolding in the core region and partial openings of the native state, determine the observed exchange rates. These findings are analyzed with respect to the amyloidogenic properties of the protein.

NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1.

EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated (15)N, (13)C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded beta sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor alpha4beta1.

Molecular models for intrastrand DNA G-quadruplexes.

BACKGROUND: Independent surveys of human gene promoter regions have demonstrated an overrepresentation of G(3)X(n1)G3X(n2)G(3)X(n3)G(3) motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data. RESULTS: We have used all quadruplex DNA structures deposited in the Nucleic Acid Database in order to select a list of fragments entailing a strand of three adjacent G's paired with another strand of three adjacent G's separated by a loop of one to four residues. These fragments were further clustered and representative fragments were finally selected. Further fragments were generated by assemblying the two strands of each fragment with loops from different fragments whenever the anchor G's were superimposable. The fragments were used to assemble G quadruplex based on a superimposability criterion. CONCLUSION: Molecular models have been generated for a large number of G(3)X(n1)G(3)X(n2)G3X(n3)G(3) sequences. For a given sequence not all topologies are possible with the available repertoire of fragments due to steric hindrance and low superimposability. Since all molecular models are generated by fragments coming from observed quadruplex structures, molecular models are in principle reliable and may be used for interpretation of experimental data. Some examples of applications are given.

Helix mobility and recognition function of the rat thyroid transcription factor 1 homeodomain - hints from 15N-NMR relaxation studies.

The backbone dynamics of the (15)N-labeled homeodomain of the rat thyroid transcription factor 1 has been studied by 2D NMR spectroscopy. Longitudinal (R(1)) and transverse (R(2)) (15)N relaxation rate constants and steady-state {(1)H}-(15)N NOEs were measured at 11.7 T. These data were analyzed by both the model-free formalism and the reduced spectral density mapping (RSDM) approaches. The global rotational correlation time, tau(m), of the thyroid transcription factor 1 homeodomain in aqueous solution at 286 K was found to be 10.51 +/- 0.05 ns by model-free formalism and 9.85 +/- 1.79 ns by RSDM calculation. The homogeneity of the values of the overall correlation time calculated from the individual (R(2)/R(1)) ratios suggested a good degree of isotropy of the global molecular motion, consistent with the similar global tau(m) results obtained with the two different methods. Tyr25 was found to undergo slow conformational exchange by both methods, whereas this contribution was identified also for Lys21, Gln22, Ile38 and His52 only by RSDM. With both methods, the C-terminal fragment of helix III was found to be more flexible than the preceding N-terminal portion, with slightly different limits between rigid and mobile moieties. Additionally, Arg53 appeared to be characterized by an intermediate motional freedom between the very flexible N-terminal and C-terminal residues and the structured core of the molecule, suggesting the occurrence of a hinge point. Finally, slow-time-scale motions observed at the end of helix I, at the end of helix II and within helix III appear to be consistent with typical fraying transitions at helical C-termini.

Molecular dynamics simulation suggests possible interaction patterns at early steps of beta2-microglobulin aggregation.

Early events in aggregation of proteins are not easily accessible by experiments. In this work, we perform a 5-ns molecular dynamics simulation of an ensemble of 27 copies of beta(2)-microglobulin in explicit solvent. During the simulation, the formation of intermolecular contacts is observed. The simulation highlights the importance of apical residues and, in particular, of those at the N-terminus end of the molecule. The most frequently found pattern of interaction involves a head-to-head contact arrangement of molecules. Hydrophobic contacts appear to be important for the establishment of long-lived (on the simulation timescale) contacts. Although early events on the pathway to aggregation and fibril formation are not directly related to the end-state of the process, which is reached on a much longer timescale, simulation results are consistent with experimental data and in general with a parallel arrangement of intermolecular beta-strand pairs.

Scoring predictive models using a reduced representation of proteins: model and energy definition.

BACKGROUND: Reduced representations of proteins have been playing a keyrole in the study of protein folding. Many such models are available, with different representation detail. Although the usefulness of many such models for structural bioinformatics applications has been demonstrated in recent years, there are few intermediate resolution models endowed with an energy model capable, for instance, of detecting native or native-like structures among decoy sets. The aim of the present work is to provide a discrete empirical potential for a reduced protein model termed here PC2CA, because it employs a PseudoCovalent structure with only 2 Centers of interactions per Amino acid, suitable for protein model quality assessment. RESULTS: All protein structures in the set top500H have been converted in reduced form. The distribution of pseudobonds, pseudoangle, pseudodihedrals and distances between centers of interactions have been converted into potentials of mean force. A suitable reference distribution has been defined for non-bonded interactions which takes into account excluded volume effects and protein finite size. The correlation between adjacent main chain pseudodihedrals has been converted in an additional energetic term which is able to account for cooperative effects in secondary structure elements. Local energy surface exploration is performed in order to increase the robustness of the energy function. CONCLUSION: The model and the energy definition proposed have been tested on all the multiple decoys' sets in the Decoys'R'us database. The energetic model is able to recognize, for almost all sets, native-like structures (RMSD less than 2.0 A). These results and those obtained in the blind CASP7 quality assessment experiment suggest that the model compares well with scoring potentials with finer granularity and could be useful for fast exploration of conformational space. Parameters are available at the url: http://www.dstb.uniud.it/~ffogolari/download/.

Solution structure of beta(2)-microglobulin and insights into fibrillogenesis.

The solution structure of human beta(2)-microglobulin (beta(2)-m) was determined by (1)H NMR spectroscopy and restrained modeling calculations. Compared to the crystal structure of type I major histocompatibility complex (MHC-I), where the protein is associated to the heavy-chain component, several differences are observed, i.e., increased separation between strands A and B, displacements of strand C' and loop DE, shortening of strands D and E. These modifications can be considered as the prodromes of the amyloid transition. Even minor charge changes in response to pH, as is the case with H31 imidazole protonation, trigger the transition that starts with unpairing of strand A. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu(2+) binding which is shown to occur primarily at H31. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches via surface charge cluster. Mutants or truncated forms of beta(2)-m can be designed to remove the instability from H31 titration or to enhance the instability through surface charge suppression. By monitoring the conformational evolution of wild-type protein and variants thereof, either in response or absence of external perturbation, valuable insights into intermediate structure and fibrillogenesis mechanisms are gained.

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus.

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same betaalphabetabetaalphabeta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8 degrees C and an unfolding free energy, DeltaG(U-F)H2O, at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.

Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature.

Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved beta2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine beta2-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type beta2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E beta-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered beta2-microglobulin molecules. This is the first single-residue resolution study of a specific beta2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in beta2-microglobulin amyloidogenicity in vivo.

Beta2-microglobulin isoforms display an heterogeneous affinity for type I collagen.

It has been claimed that beta2-microglobulin (beta2-m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the beta2-m amyloid deposits that target the osteo-articular system. The binding parameters of the interaction between collagen and beta2-m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of beta2-m. Wild-type beta2-m binds collagen type I with a Kd of 4.1 x 10(-4) M and type II with 2.3 x 10(-3) M. By the BIAcore system we monitored the binding properties of the conformers of the slow phase of folding of beta2-m. The folding intermediates during the slow phase of folding do not display any significant difference with respect to the binding properties of the fully folded molecule. The affinity of beta2-m truncated at the third N-terminal residue does not differ from that reported for the wild-type protein. Increased affinity for collagen type I is found in the case of N-terminal truncated species lacking of six residues. The Kd of this species is 3.4 x 10 (-5) M at pH 7.4 and its affinity increases to 4.9 x 10(-6) M at pH 6.4. Fluctuations of the affinity caused by beta2-m truncation and pH change can cause modifications of protein concentration in the solvent that surrounds the collagen, and could contribute to generate locally a critical protein concentration able to prime the protein aggregation.

The solution structure of human beta2-microglobulin reveals the prodromes of its amyloid transition.

The solution structure of human beta2-microglobulin (beta2-m), the nonpolymorphic component of class I major histocompatibility complex (MHC-I), was determined by (1)H NMR spectroscopy and restrained modeling calculations. Compared to previous structural data obtained from the NMR secondary structure of the isolated protein and the crystal structure of MHC-I, in which the protein is associated to the heavy-chain component, several differences are observed. The most important rearrangements were observed for (1) strands V and VI (loss of the C-terminal and N-terminal end, respectively), (2) interstrand loop V-VI, and (3) strand I, including the N-terminal segment (displacement outward of the molecular core). These modifications can be considered as the prodromes of the amyloid transition. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches at the interface with heavy chain and the nearby region at the surface charge cluster of the C-terminal segment. As a result, the molecule is placed in a state in which even minor charge and solvation changes in response to pH or ionic-strength variations can easily compromise the hydrophobic/hydrophilic balance and trigger the transition into a partially unfolded intermediate that starts with unpairing of strand I and leads to polymerization and precipitation into fibrils or amorphous aggregates. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu(2+) binding, which is shown to occur primarily at His 31 and involve partially also His 13, the next available His residue along the partial unfolding pathway.

Fast structure similarity searches among protein models: efficient clustering of protein fragments.

BACKGROUND: For many predictive applications a large number of models is generated and later clustered in subsets based on structure similarity. In most clustering algorithms an all-vs-all root mean square deviation (RMSD) comparison is performed. Most of the time is typically spent on comparison of non-similar structures. For sets with more than, say, 10,000 models this procedure is very time-consuming and alternative faster algorithms, restricting comparisons only to most similar structures would be useful. RESULTS: We exploit the inverse triangle inequality on the RMSD between two structures given the RMSDs with a third structure. The lower bound on RMSD may be used, when restricting the search of similarity to a reasonably low RMSD threshold value, to speed up similarity searches significantly. Tests are performed on large sets of decoys which are widely used as test cases for predictive methods, with a speed-up of up to 100 times with respect to all-vs-all comparison depending on the set and parameters used. Sample applications are shown. CONCLUSIONS: The algorithm presented here allows fast comparison of large data sets of structures with limited memory requirements. As an example of application we present clustering of more than 100000 fragments of length 5 from the top500H dataset into few hundred representative fragments. A more realistic scenario is provided by the search of similarity within the very large decoy sets used for the tests. Other applications regard filtering nearly-indentical conformation in selected CASP9 datasets and clustering molecular dynamics snapshots. AVAILABILITY: A linux executable and a Perl script with examples are given in the supplementary material (Additional file 1). The source code is available upon request from the authors.