Interstrand Interactions Research Articles

Accessing Three-Dimensional Crystals with Incorporated Guests through Metal-Directed Coiled-Coil Peptide Assembly.

Obtaining three-dimensional (3D) protein and peptide crystals on demand requires a precisely orchestrated hierarchical assembly of biopolymer building blocks. In this work, we disclose a metal-ion-mediated strategy to assemble trimeric coiled-coil peptides in a head-to-tail fashion into linear strands with interstrand interactions. This design led to hexagonal 3D peptide crystal formation within 30 min in the presence of divalent metal ions. The crystal morphology could be controlled by varying the metal ion/peptide ratio, resulting in hexagonal discs to rods. Diffraction studies elucidated the head-to-tail arrangement of the coiled-coil linear strands and their hexagonal, antiparallel packing within the crystal. Unsatisfied ligands at the hexagonal ends of the crystals were harnessed as a powerful means to direct His-tagged fluorophores to distinct locations within the crystals. Overall, the designed hierarchical assembly provides a facile means to obtain 3D peptide crystals and incorporate His-tag-based cargoes and may have potential use in drug delivery and sensor design.

Stabilization of i-motif structures by 2'-β-fluorination of DNA.

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH+). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands. To avoid this limitation and expand the range of environmental conditions supporting i-motif folding, we replaced the sugar in DNA by 2-deoxy-2-fluoroarabinose. We demonstrate that such a modification significantly stabilizes i-motif formation over a wide pH range, including pH 7. Nuclear magnetic resonance experiments reveal that 2-deoxy-2-fluoroarabinose adopts a C2′-endo conformation, instead of the C3′-endo conformation usually found in unmodified i-motifs. Nevertheless, this substitution does not alter the overall i-motif structure. This conformational change, together with the changes in charge distribution in the sugar caused by the electronegative fluorine atoms, leads to a number of favorable sequential and inter-strand electrostatic interactions. The availability of folded i-motifs at neutral pH will aid investigations into the biological function of i-motifs in vitro, and will expand i-motif applications in nanotechnology.

Fmoc-RGDS based fibrils: atomistic details of their hierarchical assembly.

We describe the 3D supramolecular structure of Fmoc-RGDS fibrils, where Fmoc and RGDS refer to the hydrophobic N-(fluorenyl-9-methoxycarbonyl) group and the hydrophilic Arg-Gly-Asp-Ser peptide sequence, respectively. For this purpose, we performed atomistic all-atom molecular dynamics simulations of a wide variety of packing modes derived from both parallel and antiparallel β-sheet configurations. The proposed model, which closely resembles the cross-β core structure of amyloids, is stabilized by π-π stacking interactions between hydrophobic Fmoc groups. More specifically, in this organization, the Fmoc-groups of β-strands belonging to the same β-sheet form columns of π-stacked aromatic rings arranged in a parallel fashion. Eight of such columns pack laterally forming a compact and dense hydrophobic core, in which two central columns are surrounded by three adjacent columns on each side. In addition to such Fmoc···Fmoc interactions, the hierarchical assembly of the constituent β-strands involves a rich variety of intra- and inter-strand interactions. Accordingly, hydrogen bonding, salt bridges and π-π stacking interactions coexist in the highly ordered packing network proposed for the Fmoc-RGDS amphiphile. Quantum mechanical calculations, which have been performed to quantify the above referred interactions, confirm the decisive role played by the π-π stacking interactions between the rings of the Fmoc groups, even though both inter-strand and intra-strand hydrogen bonds and salt bridges also play a non-negligible role. Overall, these results provide a solid reference to complement the available experimental data, which are not precise enough to determine the fibril structure, and reconcile previous independent observations.

The structure of chromophore-grafted amyloid-β(12-28) dimers in the gas-phase: FRET-experiment guided modelling.

We present theoretical modelling, ion mobility spectrometry and action-FRET experiments for chromophore-grafted amyloid-β(12-28) dimers. A first-principles global minimum search based on replica-exchange molecular dynamics (REMD) leads to a compact structure with strong interstrand interactions. We use REMD with a distance restraint that implements an adaptive effective bias upon average FRET-efficiencies and thus guides the sampling by the action-FRET measurement. This procedure leads to a pair of weakly interacting peptides. Ion-mobility confirms that the weakly interacting structure and not the global minimum with strongly interacting peptides is populated in the experiment. The presence of a high energy barrier between the two structural families, as evidenced from the MD data, suggests that a kinetically trapped structure is observed in the experiment.

Different nanostructures caused by competition of intra- and inter-β-sheet interactions in hierarchical self-assembly of short peptides

To understand how molecular interactions lead to the self-assembly of twisted, helical and flat nanoribbons, we have compared the hierarchical self-assembly processes of three selected octapeptides with the same amino acid composition but different sequences by both experiments and molecular dynamics (MD) simulations. KE-F8 (NH2-KEFFFFKE-CONH2) and EK-F8 (NH2-KEFFFFEK-CONH2) have the same distribution of hydrophobic residues and only differ by swapping the positive and negative charged residues at their C-terminals, while KFE-8 (NH2-KFEFKFEF-CONH2) differs from KE-F8 and EK-F8 by having all hydrophobic and charged residues evenly distributed. MD simulations indicated that the competition between electrostatic and hydrophobic interactions at the molecular level results in different initial packing modes: KE-F8 monomers form completely matched anti-parallel β-sheets, EK-F8 monomers align with one residue shifting, and KFE-8 monomers pack β-sheets with two heterogeneous surfaces, consistent with previously suggested models. Driven by inter-strand and inter-sheet interactions, further growth of these molecular templates leads to larger oligomers with different twisting and stacking degrees, which are structurally consistent with the experimentally observed self-assembled morphologies. Further MD simulations showed that the competition between intra-β-sheet and inter-β-sheet interactions is responsible for the different twisting and stacking degrees of β-sheets and the subsequent formation of different nanostructures (twisted ribbons for KE-F8, helical ribbons/tubes for EK-F8 and flat ribbons for KFE-8). This study thus provided an important mechanistic insight into the fine tuning of molecular packing and interactions via peptide sequence variation leading to controllable self-assembly of twisted, helical and flat nanostructures.

Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins

The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans.

Solid-To-Fluid DNA Transition Inside HSV-1 Capsid Close to the Temperature of Infection

DNA in the human Herpes simplex virus type 1 (HSV-1) capsid is packaged to a tight density. This leads to tens of atmospheres of internal pressure responsible for the delivery of the herpes genome into the cell nucleus. In this study we show that despite its liquid crystalline state inside the capsid, the DNA is fluid-like which facilitates its ejection into the cell nucleus during infection. We found that the sliding friction between closely packaged DNA strands, caused by interstrand repulsive interactions, is reduced by the ionic environment, mimicking that of epithelial cells and neurons susceptible to herpes infection. However, variations in the ionic conditions corresponding to neuronal activity can restrict DNA mobility in the capsid, making it more solid-like. This can inhibit intranuclear DNA release and interfere with viral replication. In addition, the temperature of the human host (37°C) induces a disordering transition of the encapsidated herpes genome which reduces interstrand interactions and provides genome mobility required for infection.

Elucidating pH-dependent collagen triple helix formation through interstrand hydroxyproline-glutamic acid interactions.

Here, we describe systematic explorations into the molecular basis underlying hydroxyproline-mediated interstrand interactions on the triple-helical stability of collagen-mimetic peptides containing glutamic acid residues. Our studies reveal that the triple-helical stability of these peptides relies on the existence of interstrand interactions between hydroxyprolines and glutamic acid residues that are pH dependent. These unique interactions have been used to engineer collagen peptides that form triple helices on demand through pH control.

Solid-to-fluid DNA transition inside HSV-1 capsid close to the temperature of infection.

DNA in the human Herpes simplex virus type 1 (HSV-1) capsid is packaged to a tight density. This leads to tens of atmospheres of internal pressure responsible for the delivery of the herpes genome into the cell nucleus. In this study we show that, despite its liquid crystalline state inside the capsid, the DNA is fluid-like, which facilitates its ejection into the cell nucleus during infection. We found that the sliding friction between closely packaged DNA strands, caused by interstrand repulsive interactions, is reduced by the ionic environment of epithelial cells and neurons susceptible to herpes infection. However, variations in the ionic conditions corresponding to neuronal activity can restrict DNA mobility in the capsid, making it more solid-like. This can inhibit intranuclear DNA release and interfere with viral replication. In addition, the temperature of the human host (37 °C) induces a disordering transition of the encapsidated herpes genome, which reduces interstrand interactions and provides genome mobility required for infection.

Purine biosynthetic intermediate-containing ribose-phosphate polymers as evolutionary precursors to RNA.

The RNA world hypothesis proposes that RNA once functioned as the principal genetic material and biological catalyst. However, RNA is a complex molecule made up of phosphate, ribose, and nucleobase moieties, and its evolution is unclear. Yakhnin has proposed a period of prebiotic chemical evolution prior to the advent of replication and Darwinian evolution, in which macromolecules containing polyols joined by phosphodiester linkages underwent spontaneous transesterification reactions with selection for stability. Although he proposes that the nucleobases were obtained during this stage from less stable macromolecules, the ultimate source of the nucleobases is not addressed. We propose that the purine nucleobases arose in situ from simpler precursors attached to a ribose-phosphate backbone, and that the weaker and less specific intra- and interstrand interactions between these precursors were the forerunners to the base pairing and base stacking interactions of the modern RNA nucleobases. Further, in line with Granick's hypothesis of biosynthetic pathways recapitulating evolution, we propose that these simpler precursors were the same or similar to intermediates of the modern de novo purine biosynthetic pathway. We propose that successive nucleobase precursors formed progressively stronger interactions that stabilized the ribose-phosphate polymer, and that the increased stability of the parent polymer drove the selection and further chemical evolution of the purine nucleobases. Such interactions may have included hydrogen bonding between ribose hydroxyls, hydrogen bonding between carbonyl oxygens and protonated amine side groups, the intra- and interstrand coordination of metal cations, and the stacking of imidazole rings. Five of the eleven steps of the modern de novo purine biosynthetic pathway have previously been shown to have alternative nonenzymatic syntheses, while a sixth step has also been proposed to occur nonenzymatically, supporting a prebiotic origin for the pathway.

In silico design and enzymatic synthesis of functional RNA nanoparticles.

ConspectusThe use of RNAs as scaffolds for biomedical applications has several advantages compared with other existing nanomaterials. These include (i) programmability, (ii) precise control over folding and self-assembly, (iii) natural functionalities as exemplified by ribozymes, riboswitches, RNAi, editing, splicing, and inherent translation and transcription control mechanisms, (iv) biocompatibility, (v) relatively low immune response, and (vi) relatively low cost and ease of production. We have tapped into several of these properties and functionalities to construct RNA-based functional nanoparticles (RNA NPs). In several cases, the structural core and the functional components of the NPs are inherent in the same construct. This permits control over the spatial disposition of the components, intracellular availability, and precise stoichiometry.To enable the generation of RNA NPs, a pipeline is being developed. On one end, it encompasses the rational design and various computational schemes that promote design of the RNA-based nanoconstructs, ultimately producing a set of sequences consisting of RNA or RNA–DNA hybrids, which can assemble into the designed construct. On the other end of the pipeline is an experimental component, which takes the produced sequences and uses them to initialize and characterize their proper assembly and then test the resulting RNA NPs for their function and delivery in cell culture and animal models. An important aspect of this pipeline is the feedback that constantly occurs between the computational and the experimental parts, which synergizes the refinement of both the algorithmic methodologies and the experimental protocols. The utility of this approach is depicted by the several examples described in this Account (nanocubes, nanorings, and RNA–DNA hybrids). Of particular interest, from the computational viewpoint, is that in most cases, first a three-dimensional representation of the assembly is produced, and only then are algorithms applied to generate the sequences that will assemble into the designated three-dimensional construct. This is opposite to the usual practice of predicting RNA structures from a given sequence, that is, the RNA folding problem. To be considered is the generation of sequences that upon assembly have the proper intra- or interstrand interactions (or both). Of particular interest from the experimental point of view is the determination and characterization of the proper thermodynamic, kinetic, functionality, and delivery protocols. Assembly of RNA NPs from individual single-stranded RNAs can be accomplished by one-pot techniques under the proper thermal and buffer conditions or, potentially more interestingly, by the use of various RNA polymerases that can promote the formation of RNA NPs cotransciptionally from specifically designed DNA templates.Also of importance is the delivery of the RNA NPs to the cells of interest in vitro or in vivo. Nonmodified RNAs rapidly degrade in blood serum and have difficulties crossing biological membranes due to their negative charge. These problems can be overcome by using, for example, polycationic lipid-based carriers. Our work involves the use of bolaamphiphiles, which are amphipathic compounds with positively charged hydrophilic head groups at each end connected by a hydrophobic chain. We have correlated results from molecular dynamics computations with various experiments to understand the characteristics of such delivery agents.

π-Cooperativity effect on the base stacking interactions in DNA: is there a novel stabilization factor coupled with base pairing H-bonds?

The results from absolutely localized molecular orbital (ALMO)-energy decomposition analysis (EDA) and ALMO-charge transfer analysis (CTA) at M06-2X/cc-pVTZ level reveal that double-proton transfer (DPT) reactions through base pairing H-bonds have nonignorable effects on the stacking energies of dinucleotide steps, which introduces us to a novel stabilization (or destabilization) factor in the DNA duplex. Thus, intra- and inter-strand base stacking interactions are coalesced with each other mediated by H-bridged quasirings between base pairs. Changes in stacking energies of dinucleotide steps depending on the positions of H atoms are due to variations in local aromaticities of individual nucleobases, manifesting π-cooperativity effects. CT analyses show that dispersion forces in dinucleotide steps can lead to radical changes in the redox properties of nucleobases, in particular those of adenine and guanine stacked dimers in a strand. Besides Watson-Crick rules, novel base pairing rules were propounded by considering CT results. According to these, additional base pairing through π-stacks of nucleobases in dinucleotide steps does not cause any intrinsic oxidative damage to the associated nucleobases throughout DPT.

Exploring the molecular basis for the metal-mediated assembly of alginate gels

The binding of sodium and calcium ions to single and multiple poly-G decamer strands has been modelled by conducting a series of molecular dynamics simulations. Implications for metal mediated inter-strand interactions and gel assembly have been explored by systematically introducing up to three strands into each of these simulations. A particular emphasis has been placed on revealing intrinsic binding modes by an unbiased initial positioning of the metal ions. The results have revealed binding modes that provide a rationale for the observed gelling of alginate by calcium rather than sodium ions. A number of junction zones involving calcium ions have been identified that result in chain aggregation. This includes a distinctive perpendicular motif that appears to be ubiquitous in previously reported AFM images of open 3-D alginate networks. The coordination geometries of the metal ions have been characterised and the metal-mediated junctions between associated strands are described in detail.

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

Theory of equilibria of elastic 2-braids with interstrand interaction

Motivated by continuum models for DNA supercoiling we formulate a theory for equilibria of 2-braids, i.e., structures formed by two elastic rods winding around each other in continuous contact and subject to a local interstrand interaction. No assumption is made on the shape of the contact curve. The theory is developed in terms of a moving frame of directors attached to one of the strands. The other strand is tracked by including in this frame the normalised closest-approach chord connecting the two strands. The kinematic constant-distance constraint is formulated at strain level through the introduction of what we call braid strains. As a result the total potential energy involves arclength derivatives of these strains, thus giving rise to a second-order variational problem. The Euler–Lagrange equations for this problem give balance equations for the overall braid force and moment referred to the moving frame as well as differential equations that can be interpreted as effective constitutive relations encoding the effect that the second strand has on the first as the braid deforms under the action of end loads. Hard contact models are used to obtain the normal contact pressure between strands that has to be non-negative for a physically realisable solution without the need for external devices such as clamps or glue to keep the strands together. The theory is first illustrated by a number of problems that can be solved analytically and then applied to several new problems that have not hitherto been treated.

The universality of β-hairpin misfolding indicated by molecular dynamics simulations

Previous molecular dynamics simulations showed that besides the experimentally measured folded structures, several β-structured polypeptides could also have misfolded "out-of-register" structures. Through the enhanced sampling molecular dynamics simulations on a series of polypeptides using either implicit or explicit solvent model, the present study systematically investigated the universality of β-hairpin misfolding and its determinants. It was observed that the misfolding could take place for almost all polypeptides under study, especially in the presence of weak side chain hydrophobicity. Moreover, the observed misfolded structures for various polypeptides share the following common features: (1) the turn length in misfolded structure is one-residue shorter than that in folded structure; (2) the hydrophobic side chains on the two strands are pointed to the opposite directions instead of packing in the same direction to form hydrophobic core cluster in the folded structure; and (3) the misfolded structure is one-residue-shifted asymmetric β-hairpin structure. The detailed analysis suggested that the misfolding of β-hairpin is the result of the competition between the formation of the alterable turn configurations and the inter-strand hydrophobic interactions. These predictions are desired to be tested by experiments.

Influences on the stability of collagen triple-helix

Collagen forms a triple helix structure which consists of three tightly coiled polyproline II-type (PPII) strands with the repeating amino acid motif Xaa-Yaa-Gly, where Xaa and Yaa are often proline (Pro) and hydroxproline (Hyp), respectively. The thermal and mechanical stability of the collagen triple-helix is a complex balance between several factors, e.g. sterical influences of substituents, stereoelectronic effects, or interstrand interactions. On the basis of computational studies, we have analyzed the influence of these effects and demonstrate that even comparatively small effects, like the stereoelectronic gauche-effect, and intramolecular hydrogen-bonding can have an impact on the triple-helix stability. In addition, our results suggest that hydrogen bond-like interactions perpendicular to the helix axis increase the stiffness of collagen. Our studies provide a deeper insight into the structure-directing factors in collagenous materials and will help to improve the synthesis of collagen-like biomaterials.

DNA hybridization kinetics: zippering, internal displacement and sequence dependence

Although the thermodynamics of DNA hybridization is generally well established, the kinetics of this classic transition is less well understood. Providing such understanding has new urgency because DNA nanotechnology often depends critically on binding rates. Here, we explore DNA oligomer hybridization kinetics using a coarse-grained model. Strand association proceeds through a complex set of intermediate states, with successful binding events initiated by a few metastable base-pairing interactions, followed by zippering of the remaining bonds. But despite reasonably strong interstrand interactions, initial contacts frequently dissociate because typical configurations in which they form differ from typical states of similar enthalpy in the double-stranded equilibrium ensemble. Initial contacts must be stabilized by two or three base pairs before full zippering is likely, resulting in negative effective activation enthalpies. Non-Arrhenius behavior arises because the number of base pairs required for nucleation increases with temperature. In addition, we observe two alternative pathways—pseudoknot and inchworm internal displacement—through which misaligned duplexes can rearrange to form duplexes. These pathways accelerate hybridization. Our results explain why experimentally observed association rates of GC-rich oligomers are higher than rates of AT- rich equivalents, and more generally demonstrate how association rates can be modulated by sequence choice.

Statistical Mechanical Model for the Transfer Free Energy of Amino Acids in the Context of Membrane Protein OmpLA

Transfer free energy scale of amino acids is fundamental for understanding membrane protein folding and for predicting membrane protein structures. Several experimental studies have been carried out to measure the free energy of partitioning amino acids to artificial lipid bilayer or biological membranes in the context of model peptides. A recent study measured water-to-bilayer transfer free energy in the context of a native β-barrel transmembrane protein OmpLA[1]. Here we report the development of a computational free energy scale based on an empirical potential energy function and statistical mechanical model. Our energy function incorporates the energy contribution of single-body burial, inter-strand interaction and sequential nearest neighbor contact interaction. Using a statistical mechanical model with a reduced state space, we computed the full partition function of OmpLA and the relative insertion free energy of amino acids replacing Ala210. The computed relative free energy scale correlates well with experimental data (r2 =0.79 with water-to-bilayer scale[1] and r2=0.88 with translocon scale[2]). In addition, depth dependency profiles of Arg and Leu are in excellent agreement with those measured by Moon and Flemming [1]. We also obtained depth dependency profiles of all 20 amino acids which provide insight to the folding process of membrane protein. Furthermore, our model showed strong coopperativity of Arg-Arg, consistent with experimental founds[1]. We further predicted that Lys-Lys show significant cooperativity, which is subject to future experimental verification.1. Moon CP, Fleming KG (2011) Side-chain hydrophobicity scale derived from transmembrane protein folding into lipid bilayers. Proc Natl Acad Sci USA108:10174-101772. Hessa, T, Kim, H, Bihlmaier, K, Lundin, C, Boekel, J, Andersson, H, Nilsson, I, White, SH, von Heijne, G (2005). Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature, 433, 7024:377-81.

Structural origin of polymorphism of Alzheimer's amyloid β-fibrils

Formation of senile plaques containing amyloid fibrils of Aβ (amyloid β-peptide) is a pathological hallmark of Alzheimer's disease. Unlike globular proteins, which fold into unique structures, the fibrils of Aβ and other amyloid proteins often contain multiple polymorphs. Polymorphism of amyloid fibrils leads to different toxicity in amyloid diseases and may be the basis for prion strains, but the structural origin for fibril polymorphism is still elusive. In the present study we investigate the structural origin of two major fibril polymorphs of Aβ40: an untwisted polymorph formed under agitated conditions and a twisted polymorph formed under quiescent conditions. Using electron paramagnetic resonance spectroscopy, we studied the inter-strand side-chain interactions at 14 spin-labelled positions in the Aβ40 sequence. The results of the present study show that the agitated fibrils have stronger inter-strand spin-spin interactions at most of the residue positions investigated. The two hydrophobic regions at residues 17-20 and 31-36 have the strongest interactions in agitated fibrils. Distance estimates on the basis of the spin exchange frequencies suggest that inter-strand distances at residues 17, 20, 32, 34 and 36 in agitated fibrils are approximately 0.2 Å (1 Å=0.1 nm) closer than in quiescent fibrils. We propose that the strength of inter-strand side-chain interactions determines the degree of β-sheet twist, which then leads to the different association patterns between different cross β-units and thus distinct fibril morphologies. Therefore the inter-strand side-chain interaction may be a structural origin for fibril polymorphism in Aβ and other amyloid proteins.