Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

The solution structure and stability of N-terminally truncated b2-microglobulin~DN6b2-m!, the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that DN6b2-m has a free energy of stabilization that is reduced by 2.5 kcal0mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at mM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that DN6b2-m is significantly less protected than its wild-type counterpart. Analysis of DN6b2-m by NMR shows that this loss of protection occurs in b strands I, III, and part of II. At mM concentration gel filtration analysis shows that DN6b2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of b2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that DN6b2-m could be a key intermediate of a proteolytic pathway of b2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of b2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between b-strands far removed from this constrain is greatly perturbed.Keywords: amyloidosis; b2-microglobulin; hydrogen exchange mass spectrometry; limited proteolysis; NMR; protein folding Amyloidoses are diseases caused by tissue deposition of protein aggregate organized in an ordered b-sheet structure. The conversion of globular proteins to insoluble fibrillar aggregates requires significant conformational changes, such as the loss of tertiary and quaternary interactions or conversion of a to b secondary structurẽ Sunde & Blake, 1998!. Of the 17 or so proteins implicated in amyloidoses the fibril morphology is indistinguishable and there does not appear to be any common features that link the soluble precursor proteins. For many of these proteins, the amyloid fibril formation is facilitated by amino acid mutations that destabilize the native state and confer a structural flexibility to the molecule, but other proteins like IAPP, wild-type TTR, and b2-microglobulin

We describe a kindred with slowly progressive gastrointestinal symptoms and autonomic neuropathy caused by autosomal dominant, hereditary systemic amyloidosis. The amyloid consists of Asp76Asn variant β(2)-microglobulin. Unlike patients with dialysis-related amyloidosis caused by sustained high plasma concentrations of wild-type β(2)-microglobulin, the affected members of this kindred had normal renal function and normal circulating β(2)-microglobulin values. The Asp76Asn β(2)-microglobulin variant was thermodynamically unstable and remarkably fibrillogenic in vitro under physiological conditions. Previous studies of β(2)-microglobulin aggregation have not shown such amyloidogenicity for single-residue substitutions. Comprehensive biophysical characterization of the β(2)-microglobulin variant, including its 1.40-Å, three-dimensional structure, should allow further elucidation of fibrillogenesis and protein misfolding.

Apolipoprotein A-I, the major structural apolipoprotein of high-density lipoproteins, efficiently protects humans from cholesterol accumulation in tissues; however, it can cause systemic amyloidosis in the presence of peculiar amino acid replacements. The wild-type molecule also has an intrinsic tendency to generate amyloid fibrils that localise within the atherosclerotic plaques. The structure, folding and metabolism of normal apolipoprotein A-I are extremely complex and as yet not completely clarified, but their understanding appears essential for the elucidation of the amyloid transition. We reviewed present knowledge on the structure, function and amyloidogenic propensity of apolipoprotein A-I with the aim of highlighting the possible molecular mechanisms that might contribute to the pathogenesis of this disease. Important clues on apolipoprotein A-I amyloidogenesis may be obtained from classical comparative studies of the properties of the wild-type versus the amyloidogenic counterpart. Additionally, in the case of apoA-I, further insights on the molecular mechanisms underlying its amyloidogenic propensity may derive from comparative studies between amyloidogenic variants and other mutations associated with hypoalphalipoproteinemia without amyloidosis.

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The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224-232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wildtype hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.misfolding | protein aggregation

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils of ␤ 2 -microglobulin (␤ 2 -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, ␤ 2 -m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology of ␤ 2 -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 ␤ 2 -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 ␤ 2 -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 ␤ 2 -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 ␤ 2 -m, that decay on moving away from the fiber surface.The deposition of ␤ 2 -microglobulin (␤ 2 -m) 2 into amyloid fibrils is the hallmark of dialysis-related amyloidosis (DRA), a disease arising as a complication of long-term hemodialysis. ␤ 2 -m is a 99-residue protein (molecular mass 11.7 kDa) that represents the light chain of the major histocompatibility complex class I (MHCI), an integral membrane protein involved in the immune response. As a result of normal MHCI catabolism, ␤ 2 -m is released in the serum from the cell surface and carried to the kidney for clearance. In the presence of kidney failure, the concentration of free circulating ␤ 2 -m can increase by up to 50-fold; the persistent increase in ␤ 2 -m concentration results in amyloid deposition, preferentially localized in the musculoskeletal system. The accumulation of ␤ 2 -m deposits has been shown to cause arthralgias, destructive osteoarthropathies, and carpal tunnel syndrome (1). Although a high concentration of ␤ 2 -m is a necessary condition for the onset of the disease, there is not a strict correlation between the disease severity and ␤ 2 -m levels (2), suggesting that other factors might be involved in ␤ 2 -m amyloid deposition.The aggregation process of ␤ 2 -m has been the object of extensive investigation for many years. Severa...

Background: We recently discovered the first natural human β2-microglobulin variant, D76N, as an amyloidogenic protein.Results: Fluid flow on hydrophobic surfaces triggers its amyloid fibrillogenesis. The α-crystallin chaperone inhibits variant-mediated co-aggregation of wild type β2-microglobulin.Conclusion: These mechanisms likely reflect in vivo amyloidogenesis by globular proteins in general.Significance: Our results elucidate the molecular pathophysiology of amyloid deposition.