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The amyloid pathology associated with long-term haemodialysis is due to the deposition of β2-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 15N NMR relaxation analysis is presented for wild-type human β2-microglobulin and W60G β2-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. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Renal failure impairs the clearance of β2-microglobulin from the serum, with the result that this protein accumulates in joints under the form of amyloid fibrils. While the molecular mechanism leading to deposition of amyloid in vivo is not totally understood, some organic compounds, such as trifluoroethanol (TFE), are commonly used to promote the elongation of amyloid fibrils in vitro. This article gives some insights into the structural properties and the conformational states of β2-microglobulin in the presence of TFE, using both the wild-type protein and the mutant Trp60Gly. The structure of the native state of the protein is rather insensitive to the presence of the alcohol, but the stability of this state is lowered in comparison to some other conformational states. In particular, a native-like folding intermediate is observed in the presence of moderate concentrations of TFE. Instead, at higher concentrations of the alcohol, the population of a disordered native-unlike state is dominant and correlates with the ability to elongate fibrils. [Copyright &y& Elsevier]

β2-microglobulin (β2m), 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 β2m 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 β2m 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 β2m 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 β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, 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 β2m mutant. [ABSTRACT FROM AUTHOR]

The backbone dynamics of the 15N-labeled homeodomain of the rat thyroid transcription factor 1 has been studied by 2D NMR spectroscopy. Longitudinal ( R1) and transverse ( R2) 15N relaxation rate constants and steady-state {1H}–15N 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, τm, 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 ( R2/ R1) ratios suggested a good degree of isotropy of the global molecular motion, consistent with the similar global τm 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. [ABSTRACT FROM AUTHOR]

Cleavage of the small amyloidogenic protein β2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related β2-microglobulin amyloidosis, since elevated levels of cleaved β2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved β2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine β2-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 β2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type β2-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 β2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E β-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 β2-microglobulin molecules. This is the first single-residue resolution study of a specific β2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in β2-microglobulin amyloidogenicity in vivo. [ABSTRACT FROM AUTHOR]

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by 1H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 Å 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 βαββαβ 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°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°C and an unfolding free energy, ΔG [ABSTRACT FROM AUTHOR]

Abstract: The solution structure of human β2-microglobulin (β2-m) was determined by 1H 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 Cu2+ 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 β2-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. [Copyright &y& Elsevier]

Three variants of human β2-microglobulin (β2-m) were compared with wild-type protein. For two variants, namely the mutant R3Aβ2-m and the form devoid of the N-terminal tripeptide (&Delta3β2-m), a reduced unfolding free energy was measured compared with wild-type β2-m, whereas an increased stability was observed for the mutant H31Yβ2-m. The solution structure could be determined by 1H NMR spectroscopy and restrained modeling only for R3Aβ2-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Yβ2-m and ΔN3β-m. Precipitation and unfolding were observed over time periods shorter than 4–6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for β2-m fibrillogenesis. The mechanism is consistent with the previous and present results on β2-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer. [ABSTRACT FROM AUTHOR]

The tripeptide glutathione (γ-L-Cys-Gly, GSH) is an important intracellular reducing agent for Cu(II) and complexation agent for Cu(I). We have studied the complexation of Cu(I) to GSH in aqueous solution at a range of pH values and Cu(I): GSH molar ratios by ¹H-NMR and 13C-NMR spectroscopy and X-ray absorption spectroscopy. The NMR data are consistent with formation of a complex with approximate 1:1 stoichiometry [Cu(SG)] as the major species with onlytholate sulfur of GSH binding to Cu(1). The rate of exchange of GSH with GS-Cu was determined to be 13 s-1 at 283 K, pH 6.8. X-ray absorption spectroscopic measurements showed that Cu(1) is coordinated to 3.1±0.3 sulfur atoms at approximately 0.222 nm in solutions (and solids ) containing GSH;Cu in 1:1 and 2:1 mol ratios. The possible structures polymeric Cu(I) -glutathione complexes are discussed. The high thermodynamic stability of Cu(I)-S bonds in Cu(I)-glutathione complexes coupled with their kinetic lability may provide efficient and specific pathways for the transport of copper in cells. [ABSTRACT FROM AUTHOR]

Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of β2-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type β2-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The β2-microglobulin -binding nanobody, Nb24, more potently inhibits D76N β2-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In β2-microglobulin knock out mice, the D76N β2-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type β2-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis. [ABSTRACT FROM AUTHOR]

The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop. [ABSTRACT FROM AUTHOR]

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications. [ABSTRACT FROM AUTHOR]

Background: The identification of reliable markers for diagnosis of breast cancer has been thoroughly addressed by metabolic profiling using nuclear magnetic resonance (NMR) spectroscopy or imaging. Several clear diagnostic indicators have emerged using either in vitro analysis of tissue extracts, ex vivo analysis of biopsies or in vivo direct spectral observations. Most of the breast cancer characteristic metabolites could be assayed by mass spectrometry (MS) to exploit the superior sensitivity of this technique and therefore reduce the traumatic impact of current biopsy procedures. Methods: Following extraction, aqueous metabolite mixtures were obtained that were submitted to liquid-chromatography, electrospray-ionization, mass spectrometry (LC/ESI-MS) analysis to estimate the content of choline (Cho) and its phosphorylated derivatives, phosphocholine (PCho) and glycerophosphocholine (GPCho). The determinations were performed using 10 samples from breast tissue biopsies, surgical specimens and one single sample of a hepatic metastasis. In addition, some measurements were also repeated using high-resolution 1H NMR spectroscopy to complement the mass spectrometry results. Results: The contents of Cho, PCho and GPCho in breast tissue extracts were estimated by LC/ESI-MS based on standard compound calibration curves. Sharply increased ratios of phosphorylated-to-unphosphorylated metabolites, PCho/ Cho and (PCho+GPCho)/Cho, were observed in all tumor samples, although without discrimination between benign and malignant lesions, contrary to samples from healthy individuals and from those with fibrocystic disease. Conclusions: The assessment of breast cancer markers by LC/ESI-MS is feasible and diagnostically valuable. In addition to high sensitivity, the approach also shows a resolution advantage for assaying choline derivatives compared to NMR, and could complement the latter. [ABSTRACT FROM AUTHOR]

The extraceUular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-termlnal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded β-sandwich fold of the gC1q domain is reduced to nine β strands with a consequent increase in the size of the central cavitylumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the α4β1 integrin. Using both Jurkat T and EA.hy926 endothelial ceils as well as site-directed mutagenesis, we demonstrate that the ability of α4β1 integrins to recognize the trimeric EMIL1N1 gC1q domain mainly depends on a single glutamic acid residue (Glu933). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILINI gC1q-α4β1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site. [ABSTRACT FROM AUTHOR]

Abstract: Amyloidosis associated to hemodialysis is caused by persistently high β2-microglobulin (β2m) serum levels. β2m is an intrinsically amyloidogenic protein whose capacity to assemble into amyloid fibrils in vitro and in vivo is concentration dependent; no β2m genetic variant is known in the human population. We investigated the roles of two evolutionary conserved Trp residues in relation to β2m structure, function and folding/misfolding by means of a combined biophysical and functional approach. We show that Trp60 plays a functional role in promoting the association of β2m in class I major histocompatibility complex; it is exposed to the solvent at the apex of a protein loop in order to accomplish such function. The Trp60→Gly mutation has a threefold effect: it stabilizes β2m, inhibits β2m amyloidogenic propensity and weakens the interaction with the class I major histocompatibility complex heavy chain. On the contrary, Trp95 is buried in the β2m core; the Trp95→Gly mutation destabilizes the protein, which is unfolded in solution, yielding nonfibrillar β2m aggregates. Trp60 and Trp95 therefore play differential and complementary roles in β2m, being relevant for function (Trp60) and for maintenance of a properly folded structure (Trp95) while affecting in distinct ways the intrinsic propensity of wild-type β2m towards self-aggregation into amyloid fibrils. [Copyright &y& Elsevier]

Systemic amyloidosis caused by extracellular deposition of insoluble fibrils derived from the pathological aggregation of circulating proteins, such as transthyretin, is a severe and usually fatal condition. Elucidation of the molecular pathogenic mechanism of the disease and discovery of effective therapies still represents a challenging medical issue. The in vitro preparation of amyloid fibrils that exhibit structural and biochemical properties closely similar to those of natural fibrils is central to improving our understanding of the biophysical basis of amyloid formation in vivo and may offer an important tool for drug discovery. Here, we compared the morphology and thermodynamic stability of natural transthyretin fibrils with those of fibrils generated in vitro either using the common acidification procedure or primed by limited selective cleavage by plasmin. The free energies for fibril formation were -12.36, -8.10, and -10.61 kcal mol-1, respectively. The fibrils generated via plasmin cleavage were more stable than those prepared at low pH and were thermodynamically and morphologically similar to natural fibrils extracted from human amyloidotic tissue. Determination of thermodynamic stability is an important tool that is complementary to other methods of structural comparison between ex vivo fibrils and fibrils generated in vitro. Our finding that fibrils created via an in vitro amyloidogenic pathway are structurally similar to ex vivo human amyloid fibrils does not necessarily establish that the fibrillogenic pathway is the same for both, but it narrows the current knowledge gap between in vitro models and in vivo pathophysiology. [ABSTRACT FROM AUTHOR]

Motivation: Electrostatic calculations are an important tool for deciphering many functional mechanisms in proteins. Generalized Born (GB) models offer a fast and convenient computational approximation over other implicit solvent-based electrostatic models. Here we present a novel GB-based web server, using the program Bluues, to calculate numerous electrostatic features including pKa-values and surface potentials. The output is organized allowing both experts and beginners to rapidly sift the data. A novel feature of the Bluues server is that it explicitly allows to find electrostatic differences between wild-type and mutant structures.Availability: The Bluues server, examples and extensive help files are available for non-commercial use at URL: http://protein.bio.unipd.it/bluues/.Contact: silvio.tosatto@unipd.it [ABSTRACT FROM AUTHOR]

Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β2-microglobulin (β2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition. [ABSTRACT FROM AUTHOR]

Neuroglobin is a recently discovered member of the globin family, mainly observed in neurons and retina. Despite the low sequence identity (less than 20% over the whole sequence for the human proteins), the general fold of neuroglobin closely resembles that of myoglobin. The latter is a paradigmatic protein for folding studies, whereas much less is known about the neuroglobin folding pathway. In this work, we show how the structural features of helix F in neuroglobin and myoglobin could represent a pivotal difference in their folding pathways. Former studies widely documented that myoglobin lacks helix F in the apo form. In this study, limited proteolysis experiments on aponeuroglobin showed that helix F does not undergo proteolytic cleavage, suggesting that, also in the apo form, this helix maintains a rigid and structured conformation. To understand better the structural properties of helices F in the two proteins, we analyzed peptides encompassing helix F of neuroglobin and myoglobin in the wild-type and mutant forms. NMR and CD experiments revealed a helical conformation for neuroglobin helix F peptide, at both pH 7 and pH 2, absent in the myoglobin peptide. In particular, NMR data suggest a secondary structure stabilization effect caused by hydrophobic interactions involving Tyr88, Leu89 and Leu92. Molecular dynamics simulations performed on the apo and holo forms of the two proteins reveal the persistence of helix F in neuroglobin even in the absence of heme. Conversely myoglobin shows a higher mobility of the N-terminus of helix F on heme removal, which leads to the loss of secondary structure. [ABSTRACT FROM AUTHOR]

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils of β2-microglobulin (β2-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, β2-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology of β2-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in β2-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role in β2-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-L-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-L-lysine is dissolved in solution with β2-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as β2-m, that decay on moving away from the fiber surface. [ABSTRACT FROM AUTHOR]