Solid-state NMR detection of 14N-13C dipolar couplings between amino acid side groups provides constraints on amyloid fibril architecture

Solid‐state nuclear magnetic resonance (SSNMR) is a powerful technique for the structural analysis of amyloid fibrils. With suitable isotope labelling patterns, SSNMR can provide constraints on the secondary structure, alignment and registration of β‐strands within amyloid fibrils and identify the tertiary and quaternary contacts defining the packing of the β‐sheet layers. Detection of ¹⁴N? ¹³C dipolar couplings may provide potentially useful additional structural constraints on β‐sheet packing within amyloid fibrils but has not until now been exploited for this purpose. Here a frequency‐selective, transfer of population in double resonance SSNMR experiment is used to detect a weak ¹⁴N? ¹³C dipolar coupling in amyloid‐like fibrils of the peptide H₂N‐SNNFGAILSS‐COOH, which was uniformly ¹³C and ¹⁵N labelled across the four C‐terminal amino acids. The ¹⁴N? ¹³C interatomic distance between leucine and asparagine side groups is constrained between 2.4 and 3.8 Å, which allows current structural models of the β‐spine arrangement within the fibrils to be refined. This procedure could be useful for the general structural analysis of other proteins in condensed phases and environments, such as biological membranes. Copyright © 2011 John Wiley & Sons, Ltd.     

Amyloid formation from an α-helix peptide bundle is seeded by 3(10)-helix aggregates

Transformation of proteins and peptides to fibrillar aggregates rich in β sheets underlies many diseases, but mechanistic details of these structural transitions are poorly understood. To simulate aggregation, four equivalents of a water‐soluble, α‐helical (65 %) amphipathic peptide (AEQLLQEAEQLLQEL) were assembled in parallel on an oxazole‐containing macrocyclic scaffold. The resulting 4α‐helix bundle is monomeric and even more α helical (85 %), but it is also unstable at pH 4 and undergoes concentration‐dependent conversion to β‐sheet aggregates and amyloid fibrils. Fibrils twist and grow with time, remaining flexible like rope (>1 μm long, 5–50 nm wide) with multiple strings (2 nm), before ageing to matted fibers. At pH 7 the fibrils revert back to soluble monomeric 4α‐helix bundles. During α→β folding we were able to detect soluble 3₁₀ helices in solution by using 2D‐NMR, CD and FTIR spectroscopy. This intermediate satisfies the need for peptide elongation, from the compressed α helix to the fully extended β strand/sheet, and is driven here by 3₁₀‐helix aggregation triggered in this case by template‐promoted helical bundling and by hydrogen‐bonding glutamic acid side chains. A mechanism involving α?α₄?(3₁₀)₄?(3₁₀)_n?(β)_n?m(β)_n equilibria is plausible for this peptide and also for peptides lacking hydrogen‐bonding side chains, with unfavourable equilibria slowing the α→β conversion.     

Spectroscopic Evidence for Polymorphic Aggregates Formed by Amyloid‐β Fragments

Understanding the structure of amyloid‐β (Aβ) aggregates is a key step towards elucidating the pathology of Alzheimer’s disease. In this work, three fragments of the Aβ_1–42 protein, Aβ_1–25 (DAEFRHDSGYEVHHQKLVFFAEDVG), Aβ_25–35 (GSNKGAIIGLM), and Aβ_33–42 (GLMVGGVVIA), were synthesized, and their aggregated structures were examined by linear infrared spectroscopy in the amide‐I (mainly the C?O stretching) region. The structures of the formed aggregates were found to be both sequence and pH dependent. The results suggest that instead of forming matured fibrils, as in the case of full‐length Aβ_1–42, both Aβ_1–25 and Aβ_33–42 form a mixture of threadlike β‐sheet fibril, soluble β‐sheet oligomer, and random coil structures. The β‐sheet conformations were found to be mainly antiparallel for the former and both parallel and antiparallel for the latter. However, the Aβ_25–35 fragment was found to form assembled fibrils containing predominantly parallel β‐sheets. The conformation and morphology of the aggregates were also confirmed by circular dichroism measurements and transmission electron microscopy. Factors influencing the structures of the aggregates formed by the Aβ fragments were discussed.     

Aggregation of Beta‐Amyloid Peptides Proximal to Zwitterionic Lipid Bilayers

One of the hallmarks of Alzheimers disease is the deposition of amyloid plaques, which consist of β‐amyloid (Aβ) peptides in fibrillar states. Nonfibrillar Aβ aggregates have been considered as an important intermediate in the pathway of fibrillization, but little is known about the formation mechanism. The on‐pathway β‐sheet intermediates of Aβ₄₀ peptides can be trapped by incubating the peptides in liposomes formed by zwitterionic lipids. The aggregates of Aβ₄₀ peptides have been prepared at a peptide concentration of less than 10 μm . Solid‐state NMR spectroscopy data show that the backbone conformation of the aggregates is almost identical to that of the fibrils formed in free solution. In contrast to anionic lipids, zwitterionic lipids, which are typical of neuronal soma, did not induce any significant conformational difference in Aβ₄₀ fibrils. This liposome–Aβ system may serve as a useful model to study the fibril formation mechanism.     

Polymer physics inspired approaches for the study of the mechanical properties of amyloid fibrils

Amyloid structures constitute a class of highly ordered nanomaterials formed by insoluble protein aggregates. These aggregates are characterized by a cross‐β structural motif in which β‐sheets are oriented perpendicular to the fibril axis and bound together by a dense hydrogen bonding network. Although they have been associated with several neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, amyloid fibrils have also been found in many physiologically beneficial roles, for instance in adhesives and hormone storage. Inspired by this natural occurrence of functional amyloid, the hierarchal self‐assembly of these structures has recently been used to develop artificial biomaterials for applications in medicine and nanotechnology. In order to realize the full potential of amyloids as functional materials, it is important to understand their fundamental mechanical properties. This review explores a range of experimental strategies to determine the mechanical properties of amyloid fibrils and discusses the results in the context of polymer physics concepts. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 281–292     

Solid‐Phase Synthesis and Characterization of N‐Terminally Elongated Aβ−3–x‐Peptides

In addition to the prototypic amyloid‐β (Aβ) peptides Aβ_1–40 and Aβ_1–42, several Aβ variants differing in their amino and carboxy termini have been described. Synthetic availability of an Aβ variant is often the key to study its role under physiological or pathological conditions. Herein, we report a protocol for the efficient solid‐phase peptide synthesis of the N‐terminally elongated Aβ‐peptides Aβ_?3–38, Aβ_?3–40, and Aβ_?3–42. Biophysical characterization by NMR spectroscopy, CD spectroscopy, an aggregation assay, and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils. Immunoprecipitation, followed by mass spectrometry, indicated that Aβ_?3–38 and Aβ_?3–40 are generated by transfected cells even in the presence of a tripartite β‐site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. The elongated Aβ peptides starting at Val(?3) can be separated from N‐terminally‐truncated Aβ forms by high‐resolution isoelectric‐focusing techniques, despite virtually identical isoelectric points. The synthetic Aβ variants and the methods presented here are providing tools to advance our understanding of the potential roles of N‐terminally elongated Aβ variants in Alzheimer's disease.     

Ester Carbonyl Vibration as a Sensitive Probe of Protein Local Electric Field

The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to gaining a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non‐natural amino acids, L ‐aspartic acid 4‐methyl ester and L ‐glutamic acid 5‐methyl ester, is a convenient and sensitive probe in this regard, since its frequency correlates linearly with the local electrostatic field for both hydrogen‐bonding and non‐hydrogen‐bonding environments. We expect that the resultant frequency–electric‐field map will find use in various applications. Furthermore, we show that, when situated in a non‐hydrogen‐bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, its application to amyloid fibrils formed by Aβ_16–22 revealed that the interior of such β‐sheet assemblies has an ε value of approximately 5.6.     

Microtubule‐Binding R3 Fragment from Tau Self‐Assembles into Giant Multistranded Amyloid Ribbons

Tau protein and its fragments self‐assemble into amyloid fibrils in the presence of polyanions, such as heparin. By combining microscopy, scattering, and spectroscopy techniques, we studied the aggregation of the 26‐mer Tau‐derived peptide alone, Tau_306–327, the third repeat fragment (R3) of the microtubule‐binding domain. We show that: i) the sole Tau_306–327 can self‐assemble into amyloid fibrils without the need of aggregation‐promoting polyanions; ii) the resulting structures consist of surprisingly large, well‐ordered 2D laminated flat ribbons, with a log‐normal distribution of the lateral width, reaching the unprecedented lateral size of 350 nm and/or 45 individual protofilaments, that is, the largest amyloid laminated structures ever observed for Tau or any other amyloidogenic sequence. Our results provide insight into the molecular determinants of Tau aggregation and open new perspectives in the understanding of the assembly of amyloid fibrils and β‐sheet‐based biomaterials.     

Construction of Chemical‐Responsive Supramolecular Hydrogels from Guest‐Modified Cyclodextrins

A methodology for preparing supramolecular hydrogels from guest‐modified cyclodextrins (CDs) based on the host–guest and hydrogen‐bonding interactions of CDs is presented. Four types of modified CDs were synthesized to understand better the gelation mechanism. The 2D ROESY NMR spectrum of β‐CD‐AmTNB (Am=amino, TNB=trinitrobenzene) reveals that the TNB group was included in the β‐CD cavity. Pulsed field gradient NMR (PFG NMR) spectroscopy and AFM show that β‐CD‐AmTNB formed a supramolecular polymer in aqueous solution through head‐to‐tail stacking. Although β‐CD‐AmTNB did not produce a hydrogel due to insufficient growth of supramolecular polymers, β‐CD‐CiAmTNB (Ci=cinnamoyl) formed supramolecular fibrils through host–guest interactions. Hydrogen bonds between the cross‐linked fibrils resulted in the hydrogel, which displayed excellent chemical‐responsive properties. Gel‐to‐sol transitions occurred by adding 1‐adamantane carboxylic acid (AdCA) or urea. ¹H NMR and induced circular dichroism (ICD) spectra reveal that AdCA released the guest parts from the CD cavity and that urea acts as a denaturing agent to break the hydrogen bonds between CDs. The hydrogel was also destroyed by adding β‐CD, which acts as the competitive host to reduce the fibrils. Furthermore, the gel changed to a sol by adding methyl orange (MO) as a guest compound, but the gel reappeared upon addition of α‐CD, which is a stronger host for MO.     

Synthesis of Diacetylene‐Containing Peptide Building Blocks and Amphiphiles,Their Self‐Assembly and Topochemical Polymerization in Organic Solvents

A series of functional iodoacetylenes was prepared and converted into the corresponding diacetylene‐substituted amino acids and peptides via Pd/Cu‐promoted sp–sp carbon cross‐coupling reactions. The unsymmetrically substituted diacetylenes can be incorporated into oligopeptides without a change in the oligopeptide strand's directionality. Thus, a series of oligopeptide‐based, amphiphilic diacetylene model compounds was synthesized, and their self‐organization as well as their UV‐induced topochemical polymerizability was investigated in comparison to related polymer‐substituted macromonomers. Solution‐phase IR spectroscopy, gelation experiments, and UV spectroscopy helped to confirm that a minimum of five N‐H???O?C hydrogen‐bonding sites was required in order to obtain reliable aggregation into stable β‐sheet‐type secondary structures in organic solvents. Furthermore, the non‐equidistant spacing of these hydrogen‐bonding sites was proven to invariably lead to β‐sheets with a parallel β‐strand orientation, and the characteristic IR‐spectroscopic signatures of the latter in organic solution was identified. Scanning force micrographs of the organogels revealed that compounds with six hydrogen‐bonding sites gave rise to high aspect ratio nanoscopic fibrils with helical superstructures but, in contrast to the related macromonomers, did not lead to uniform supramolecular polymers. The UV‐induced topochemical polymerization within the β‐sheet aggregates was successful, proving parallel β‐strand orientation and highlighting the effect of the number and pattern of N‐H???O?C hydrogen‐bonding sites as well as the hydrophobic residue in the molecular structure on the formation of higher structures and reactivity.     

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid β (1–40)

A small library of rationally designed amyloid β [Aβ(1–40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aβ(1–40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid‐state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aβ(1–40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aβ fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross‐β structure.     

Induced Folding of Protein‐Sized Foldameric β‐Sandwich Models with Core β‐Amino Acid Residues

The mimicry of protein‐sized β‐sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin‐14 β‐sheet has been used as a template, and α→β residue mutations were carried out in the hydrophobic core (positions 12 and 19). β‐Residues with diverse structural properties were utilized: Homologous β³‐amino acids, (1R,2S)‐2‐aminocyclopentanecarboxylic acid (ACPC), (1R,2S)‐2‐aminocyclohexanecarboxylic acid (ACHC), (1R,2S)‐2‐aminocyclohex‐3‐enecarboxylic acid (ACEC), and (1S,2S,3R,5S)‐2‐amino‐6,6‐dimethylbicyclo[3.1.1]heptane‐3‐carboxylic acid (ABHC). Six α/β‐peptidic chains were constructed in both monomeric and disulfide‐linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced β‐sheet formation in the 64‐residue foldameric systems. Core replacement with (1R,2S)‐ACHC was found to be unique among the β‐amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen‐bonding network and to fit sterically into the hydrophobic interior of the β‐sandwich. The novel β‐sandwich model containing 25 % unnatural building blocks afforded protein‐like thermal denaturation behavior.     

Direct Nanospectroscopic Verification of the Amyloid Aggregation Pathway

The aggregation pathways of neurodegenerative peptides determine the disease etiology, and their better understanding can lead to strategies for early disease treatment. Previous research has allowed modelling of hypothetic aggregation pathways. However, their direct experimental observation has been elusive owing to methodological limitations. Herein, we demonstrate that nanoscale chemical mapping by tip‐enhanced Raman spectroscopy of single amyloid fibrils at various stages of aggregation captures the fibril formation process. We identify changes in TERS/Raman marker bands for Aβ_1‐42, including the amide III band (above 1255 cm^?1 for turns/random coil and below 1255 cm^?1 for β‐sheet conformation). The spatial distribution of β‐sheets in aggregates is determined, allowing verification of a particular fibrillogenesis pathway, starting from aggregation of monomers to meta‐stable oligomers, which then rearrange to ordered β‐sheets, already at the oligomeric or protofibrillar stage.     

Studies on the cross‐interaction between hIAPP and Aβ25–35 and the aggregation process in binary mixture by electrospray ionization‐ion mobility‐mass spectrometry

A wealth of epidemiological evidence indicates a strong link between type 2 diabetes (T2D) and Alzheimer's disease (AD). The fiber deposition with cross‐β‐sheet structure formed by self‐aggregation and misfolding of amyloidogenic peptides is a common hallmark of both diseases. For the patients with T2D, the fibrils are mainly found in the islets of Langerhans that results from the accumulation of human islet amyloid polypeptide (hIAPP). The major component of aggregates located in the brain of AD patients is amyloid‐β (Aβ). Many biophysical and physiological properties are shared by hIAPP and Aβ, and both peptides show similar cytotoxic mechanisms. Therefore, it is meaningful to investigate the possible cross‐interactions of hIAPP and Aβ in both diseases. In this article, the segment 25–35 of Aβ was selected because Aβ_25–35 was a core region in the process of amyloid formation and showed similar aggregation tendency and toxicity with full‐length Aβ. The electrospray ionization‐ion mobility‐mass spectrometry analysis and thioflavin T fluorescence kinetic analysis combined with transmission electron microscopy were used to explore the effects of the coexistence of Aβ_25–35 and hIAPP on the self‐aggregation of both peptides and whether there was co‐assembly in fibrillation. The results indicated that the aggregation of hIAPP and Aβ_25–35 had two nucleation stages in the binary mixtures. hIAPP and Aβ_25–35 had a high binding affinity and a series of hetero‐oligomers formed in the mixtures of hIAPP and Aβ_25–35 in the early stage. The cross‐reaction between hIAPP monomers and Aβ_25–35 monomers as well as a little of oligomers during primary nucleation stage could accelerate the aggregation of Aβ_25–35. However, owing to the obvious difference in aggregation ability between hIAPP and Aβ_25–35, this cross‐interaction had no significant impact on the self‐assembly of hIAPP. Our study may offer a better understanding for exploring the molecular mechanism of the association between AD and T2D observed in clinical and epidemiological studies and developing therapeutic strategies against amyloid diseases.     

Engineering a β‐Helical d,l‐Peptide for Folding in Polar Media

β Helices—helices formed by alternating d,l ‐peptides and stabilized by β‐sheet hydrogen bonding—are found naturally in only a handful of highly hydrophobic peptides. This paper explores the scope of β‐helical structure by presenting the first design and biophysical characterization of a hydrophilic d,l ‐peptide, 1 , that forms a β helix in methanol. The design of 1 is based on the β‐hairpin/β helix—a new supersecondary that had been characterized previously only for hydrophobic peptides in nonpolar solvents. Incorporating polar residues in 1 provided solubility in methanol, in which the peptide adopts the expected β‐hairpin/β‐helical structure, as evidenced by CD, analytical ultracentrifugation (AUC), NMR spectroscopy, and NMR‐based structure calculations. Upon titration with water (at constant peptide concentration), the structure in methanol ( 1 m ) transitions cooperatively to an extended conformation ( 1 w ) resembling a cyclic β‐hairpin; observation of an isodichroic point in the solvent‐dependent CD spectra indicates that this transition is a two‐state process. In contrast, neither 1 m nor 1 w show cooperative thermal melting; instead, their structures appear intact at temperatures as high as 65 °C; this observation suggests that steric constraint is dominant in stabilizing these structures. Finally, the ¹H NMR CαH spectroscopic resonances of 1 m are downfield‐shifted with respect to random‐coil values, a hitherto unreported property for β helices that appears to be a general feature of these structures. These results show for the first time that an appropriately designed β‐helical peptide can fold stably in a polar solvent; furthermore, the structural and spectroscopic data reported should prove useful in the future design and characterization of water‐soluble β helices.     

β‐Sheet to Helical‐Sheet Evolution Induced by Topochemical Polymerization: Cross‐α‐Amyloid‐like Packing in a Pseudoprotein with Gly‐Phe‐Gly Repeats

Protein‐mimics are of great interest for their structure, stability, and properties. We are interested in the synthesis of protein‐mimics containing triazole linkages as peptide‐bond surrogate by topochemical azide‐alkyne cycloaddition (TAAC) polymerization of azide‐ and alkyne‐modified peptides. The rationally designed dipeptide N₃‐CH₂CO‐Phe‐NHCH₂CCH ( 1 ) crystallized in a parallel β‐sheet arrangement and are head‐to‐tail aligned in a direction perpendicular to the β‐sheet‐direction. Upon heating, crystals of 1 underwent single‐crystal‐to‐single‐crystal polymerization forming a triazole‐linked pseudoprotein with Gly‐Phe‐Gly repeats. During TAAC polymerization, the pseudoprotein evolved as helical chains. These helical chains are laterally assembled by backbone hydrogen bonding in a direction perpendicular to the helical axis to form helical sheets. This interesting helical‐sheet orientation in the crystal resembles the cross‐α‐amyloids, where α‐helices are arranged laterally as sheets.     

A Designed Three‐Stranded β‐Sheet in an α/β Hybrid Peptide

The incorporation of β‐amino acid residues into the antiparallel β‐strand segments of a multi‐stranded β‐sheet peptide is demonstrated for a 19‐residue peptide, Boc‐LV^βFV^DPGL^βFVVL^DPGLVL^βFVV‐OMe (BBH19). Two centrally positioned ^DPro–Gly segments facilitate formation of a stable three‐stranded β‐sheet, in which β‐phenylalanine (^βPhe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR‐derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well‐defined three‐stranded β‐sheet structure in solution. Cross‐strand interactions between ^βPhe3/^βPhe17 and ^βPhe3/Val15 residues define orientations of these side‐chains. The observation of close contact distances between the side‐chains on the N‐ and C‐terminal strands of the three‐stranded β‐sheet provides strong support for the designed structure. Evidence is presented for multiple side‐chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three‐stranded β‐sheet structures, which in turn influences the conformational interconversion between type I′ and type II′ β‐turns at the two ^DPro–Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc‐LV^βFV^DPGL^βFVV‐OMe (BBH10), which has been previously characterized as a type I′ β‐turn nucleated hairpin, is shown to favour a type II′ β‐turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.     

A pH Switch for β‐Sheet Protein Folding

Protein design advancements have led to biotechnological strategies based on more stable and more specific structures. Herein we present a 6‐residue sequence (HPATGK) that acts as a stable structure‐nucleating turn at physiological and higher pH but is notably unfavorable for chain direction reversal at low pH. When placed into the turn of a β‐sheet, this leads to a pH switch of folding. Using a standard 3‐stranded β‐sheet model, the WW domain, it was found that the pH switch sequence insertion caused minimal change at pH 8 but a ca. 50 °C drop in the melting temperature (T_m) was observed at pH 2.5: ΔΔG_F ≥11.3 kJ mol⁻¹. Using the strategies demonstrated in this article, the redesign of β‐sheets to contain a global, or local, pH‐dependent conformational switch should be possible.     

A rare occurrence of a β‐turn in an amyloid βA4 peptide

The fragment β(25–35) of the amyloid β‐peptide, like its parent βA4, has shown neurotrophic and late neurotoxic activities in cultured cells. The 3D structure of this important peptide was examined by ¹H and ¹³C 2D‐NMR and MD simulations in DMSO‐d₆ and water. The NMR parameters of chemical shift, ³J(N,Hα) coupling constants, temperature coefficients of NH chemical shifts and the pattern of intra and inter‐residue NOEs were used to deduce the structures. In DMSO‐d₆, the peptide was found to take up a type I β‐turn around the C‐terminal residues Ile⁸–Gly⁹–Leu¹⁰–Met¹¹, whereas in water at pH 5.5, it adopts a random coil conformation. This is only the second report of a β‐turn in the β‐amyloid class of peptides. The solution structures generated using restrained molecular dynamics were refined by MARDIGRAS to an R factor of 0.33 in the case of DMSO‐d₆ and to 0.56 for water. Copyright © 2002 John Wiley & Sons, Ltd.     

Reduction of conformational mobility and aggregation in W60G β2‐microglobulin: assessment by 15N NMR relaxation

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.