Performance of SOBA-AD blood test in discriminating Alzheimer's disease patients from cognitively unimpaired controls in two independent cohorts.

Amyloid-beta (Aß) toxic oligomers are critical early players in the molecular pathology of Alzheimer's disease (AD). We have developed a Soluble Oligomer Binding Assay (SOBA-AD) for detection of these Aß oligomers that contain α-sheet secondary structure that discriminates plasma samples from patients on the AD continuum from non-AD controls. We tested 265 plasma samples from two independent cohorts to investigate the performance of SOBA-AD. Testing was performed at two different sites, with different personnel, reagents, and instrumentation. Across two cohorts, SOBA-AD discriminated AD patients from cognitively unimpaired (CU) subjects with 100% sensitivity, > 95% specificity, and > 98% area under the curve (AUC) (95% CI 0.95-1.00). A SOBA-AD positive readout, reflecting α-sheet toxic oligomer burden, was found in AD patients, and not in controls, providing separation of the two populations, aside from 5 SOBA-AD positive controls. Based on an earlier SOBA-AD study, the Aß oligomers detected in these CU subjects may represent preclinical cases of AD. The results presented here support the value of SOBA-AD as a promising blood-based tool for the detection and confirmation of AD.

Mechanistic insights into the role of amyloid-ß in innate immunity.

Colocalization of microbial pathogens and the ß-amyloid peptide (Aß) in the brain of Alzheimer's disease (AD) patients suggests that microbial infection may play a role in sporadic AD. Aß exhibits antimicrobial activity against numerous pathogens, supporting a potential role for Aß in the innate immune response. While mammalian amyloid is associated with disease, many bacteria form amyloid fibrils to fortify the biofilm that protects the cells from the surrounding environment. In the microbial AD hypothesis, Aß aggregates in response to infection to combat the pathogen. We hypothesize that this occurs through toxic Aß oligomers that contain α-sheet structure and form prior to fibrillization. De novo designed α-sheet peptides specifically bind to the α-sheet structure present in the oligomers of both bacterial and mammalian amyloidogenic proteins to neutralize toxicity and inhibit aggregation. Here, we measure the effect of E. coli on Aß, including upregulation, aggregation, and toxicity. Additionally, we determined the effect of Aß structure on E. coli amyloid fibrils, or curli comprised of the CsgA protein, and biofilm formation. We found that curli formation by E. coli increased Aß oligomer production, and Aß oligomers inhibited curli biogenesis and reduced biofilm cell density. Further, curli and biofilm inhibition by Aß oligomers increased E. coli susceptibility to gentamicin. Toxic oligomers of Aß and CsgA interact via α-sheet interactions, neutralizing their toxicity. These results suggest that exposure to toxic oligomers formed by microbial pathogens triggers Aß oligomer upregulation and aggregation to combat infection via selective interactions between α-sheet oligomers to neutralize toxicity of both species with subsequent inhibition of fibrillization.

Human islet amyloid polypeptide-induced ß-cell cytotoxicity is linked to formation of α-sheet structure.

Type 2 diabetes (T2D) results from insulin secretory dysfunction arising in part from the loss of pancreatic islet ß-cells. Several factors contribute to ß-cell loss, including islet amyloid formation, which is observed in over 90% of individuals with T2D. The amyloid is comprised of human islet amyloid polypeptide (hIAPP). Here we provide evidence that early in aggregation, hIAPP forms toxic oligomers prior to formation of amyloid fibrils. The toxic oligomers contain α-sheet secondary structure, a nonstandard secondary structure associated with toxic oligomers in other amyloid diseases. De novo, synthetic α-sheet compounds designed to be nontoxic and complementary to the α-sheet structure in the toxic oligomers inhibit hIAPP aggregation and neutralize oligomer-mediated cytotoxicity in cell-based assays. In vivo administration of an α-sheet design to mice for 4 weeks revealed no evidence of toxicity nor did it elicit an immune response. Furthermore, the α-sheet designs reduced endogenous islet amyloid formation and mitigation of amyloid-associated ß-cell loss in cultured islets isolated from an hIAPP transgenic mouse model of islet amyloidosis. Characterization of the involvement of α-sheet in early aggregation of hIAPP and oligomer toxicity contributes to elucidation of the molecular mechanisms underlying amyloid-associated ß-cell loss.

Designed α-sheet peptides disrupt uropathogenic E. coli biofilms rendering bacteria susceptible to antibiotics and immune cells.

Uropathogenic Escherichia coli account for the largest proportion of nosocomial infections in the United States. Nosocomial infections are a major source of increased costs and treatment complications. Many infections are biofilm associated, rendering antibiotic treatments ineffective or cause additional complications (e.g., microbiome depletion). This work presents a potentially complementary non-antibiotic strategy to fight nosocomial infections by inhibiting the formation of amyloid fibrils, a proteinaceous structural reinforcement known as curli in E. coli biofilms. Despite extensive characterization of the fibrils themselves and their associated secretion system, mechanistic details of curli assembly in vivo remain unclear. We hypothesized that, like other amyloid fibrils, curli polymerization involves a unique secondary structure termed "α-sheet". Biophysical studies herein confirmed the presence of α-sheet structure in prefibrillar species of CsgA, the major component of curli, as it aggregated. Binding of synthetic α-sheet peptides to the soluble α-sheet prefibrillar species inhibited CsgA aggregation in vitro and suppressed amyloid fibril formation in biofilms. Application of synthetic α-sheet peptides also enhanced antibiotic susceptibility and dispersed biofilm-resident bacteria for improved uptake by phagocytic cells. The ability of synthetic α-sheet peptides to reduce biofilm formation, improve antibiotic susceptibility, and enhance clearance by macrophages has broad implications for combating biofilm-associated infections.

Molecular Dynamics Methods for Antibody Design.

Complex and coordinated dynamics are closely connected with protein functions, including the binding of antibodies to antigens. Knowledge of such dynamics could improve the design of antibodies. Molecular dynamics (MD) simulations provide a "computational microscope" that can resolve atomic motions and inform antibody design efforts.

SOBA: Development and testing of a soluble oligomer binding assay for detection of amyloidogenic toxic oligomers.

The formation of toxic Amyloid ß-peptide (Aß) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aß oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aß oligomers produce strong SOBA signals, while monomeric and ß-sheet protofibrillar Aß do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aß oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for a-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia.

The role of α-sheet structure in amyloidogenesis: characterization and implications.

Amyloid diseases are linked to protein misfolding whereby the amyloidogenic protein undergoes a conformational change, aggregates and eventually forms amyloid fibrils. While the amyloid fibrils and plaques are hallmarks of these diseases, they typically form late in the disease process and do not correlate with disease. Instead, there is growing evidence that smaller, soluble toxic oligomers form prior and appear to be early triggers of the molecular pathology underlying these diseases. Nearly 20 years ago, we proposed the α-sheet hypothesis after discovering that the early conformational changes observed during atomistic molecular dynamics simulations involve the formation of a non-standard protein structure, α-sheet. Furthermore, we proposed that toxic oligomers contain α-sheet structure and that preferentially targeting this structure could neutralize the toxicity, prevent further aggregation and serve as the basis for early detection of disease. Here, we present the origin of the α-sheet hypothesis and describe α-sheet structure and the corresponding mechanisms of conversion. We discuss experimental studies demonstrating that both mammalian and bacterial amyloid systems form α-sheet oligomers before converting to conventional ß-sheet fibrils. Furthermore, we show that the process can be inhibited with de novo designed α-sheet peptides complementary to the structure in the toxic oligomers.

Modulation of post-powerstroke dynamics in myosin II by 2'-deoxy-ADP.

Muscle myosins are molecular motors that hydrolyze ATP and generate force through coordinated interactions with actin filaments, known as cross-bridge cycling. During the cross-bridge cycle, functional sites in myosin 'sense' changes in interactions with actin filaments and the nucleotide binding region, resulting in allosteric transmission of information throughout the structure. We investigated whether the dynamics of the post-powerstroke state of the cross-bridge cycle are modulated in a nucleotide-dependent fashion. We compared molecular dynamics simulations of the myosin II motor domain (M) from Dictyostelium discoideum in the presence of ADP (M.ADP) versus 2'-deoxy-ADP bound myosin (M.dADP). We found that dADP was more flexible than ADP and the two nucleotides interacted with myosin in different ways. Replacement of ADP with dADP in the post-powerstroke state also altered the conformation of the actin binding region in myosin heads. Our results provide atomic level insights into allosteric communication networks in myosin that provide insight into the nucleotide-dependent dynamics of the cross-bridge cycle.

Conserved patterns and interactions in the unfolding transition state across SH3 domain structural homologues.

Proteins with similar structures are generally assumed to arise from similar sequences. However, there are more cases than not where this is not true. The dogma is that sequence determines structure; how, then, can very different sequences fold to the same structure? Here, we employ high temperature unfolding simulations to probe the pathways and specific interactions that direct the folding and unfolding of the SH3 domain. The SH3 metafold in the Dynameomics Database consists of 753 proteins with the same structure, but varied sequences and functions. To investigate the relationship between sequence and structure, we selected 17 targets from the SH3 metafold with high sequence variability. Six unfolding simulations were performed for each target, transition states were identified, revealing two general folding/unfolding pathways at the transition state. Transition states were also expressed as mathematical graphs of connected chemical nodes, and it was found that three positions within the structure, independent of sequence, were consistently more connected within the graph than any other nearby positions in the sequence. These positions represent a hub connecting different portions of the structure. Multiple sequence alignment and covariation analyses also revealed certain positions that were more conserved due to packing constraints and stabilizing long-range contacts. This study demonstrates that members of the SH3 domain with different sequences can unfold through two main pathways, but certain characteristics are conserved regardless of the sequence or unfolding pathway. While sequence determines structure, we show that disparate sequences can provide similar interactions that influence folding and lead to similar structures.

Edge Strand Dissociation and Conformational Changes in Transthyretin under Amyloidogenic Conditions.

During amyloidogenesis, proteins undergo conformational changes that allow them to aggregate and assemble into insoluble, fibrillar structures. Soluble oligomers that form during this process typically contain 2-24 monomeric subunits and are cytotoxic. Before the formation of these soluble oligomers, monomeric species first adopt aggregation-competent conformations. Knowledge of the structures of these intermediate states is invaluable to the development of molecular strategies to arrest pathological amyloid aggregation. However, the highly dynamic and interconverting nature of amyloidogenic species limits biophysical characterization of their structures during amyloidogenesis. Here, we use molecular dynamics simulations to probe conformations sampled by monomeric transthyretin under amyloidogenic conditions. We show that certain ß-strands in transthyretin tend to unfold and sample nonnative conformations and that the edge strands in one ß-sheet (the DAGH sheet) are particularly susceptible to conformational changes in the monomeric state. We also find that changes in the tertiary structure of transthyretin can be associated with disruptions to the secondary structure. We evaluated the conformations produced by molecular dynamics by calculating how well molecular-dynamics-derived structures reproduced NMR-derived interatomic distances. Finally, we leverage our computational results to produce experimentally testable hypotheses that may aid experimental explorations of pathological conformations of transthyretin.

Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2'-deoxy-ATP.

KEY POINTS: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation. The naturally occurring nucleotide 2-deoxy-ATP (dATP) is a myosin activator that enhances cross-bridge binding and kinetics. X-ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin-binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation. This study of the dATP-induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin-targeted molecular compounds to treat muscle diseases. ABSTRACT: Here we used time-resolved small-angle X-ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first-order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6 ) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2-deoxy-ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X-ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin-binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions.

Tumorigenic p53 mutants undergo common structural disruptions including conversion to α-sheet structure.

The p53 protein is a commonly studied cancer target because of its role in tumor suppression. Unfortunately, it is susceptible to mutation-associated loss of function; approximately 50% of cancers are associated with mutations to p53, the majority of which are located in the central DNA-binding domain. Here, we report molecular dynamics simulations of wild-type (WT) p53 and 20 different mutants, including a stabilized pseudo-WT mutant. Our findings indicate that p53 mutants tend to exacerbate latent structural-disruption tendencies, or vulnerabilities, already present in the WT protein, suggesting that it may be possible to develop cancer therapies by targeting a relatively small set of structural-disruption motifs rather than a multitude of effects specific to each mutant. In addition, α-sheet secondary structure formed in almost all of the proteins. α-Sheet has been hypothesized and recently demonstrated to play a role in amyloidogenesis, and its presence in the reported p53 simulations coincides with the recent re-consideration of cancer as an amyloid disease.

Shared unfolding pathways of unrelated immunoglobulin-like ß-sandwich proteins.

The Dynameomics project contains native state and unfolding simulations of 807 protein domains, where each domain is representative of a different metafold; these metafolds encompass ~97% of protein fold space. There is a long-standing question in structural biology as to whether proteins in the same fold family share the same folding/unfolding characteristics. Using molecular dynamics simulations from the Dynameomics project, we conducted a detailed study of protein unfolding/folding pathways for 5 protein domains from the immunoglobulin (Ig)-like ß-sandwich metafold (the highest ranked metafold in our database). The domains have sequence similarities ranging from 4 to 15% and are all from different SCOP superfamilies, yet they share the same overall Ig-like topology. Despite having very different amino acid sequences, the dominant unfolding pathway is very similar for the 5 proteins, and the secondary structures that are peripheral to the aligned, shared core domain add variability to the unfolding pathway. Aligned residues in the core domain display consensus structure in the transition state primarily through conservation of hydrophobic positions. Commonalities in the obligate folding nucleus indicate that insights into the major events in the folding/unfolding of other domains from this metafold may be obtainable from unfolding simulations of a few representative proteins.

Drivers of α-Sheet Formation in Transthyretin under Amyloidogenic Conditions.

Amyloid diseases make up a set of fatal disorders in which proteins aggregate to form fibrils that deposit in tissues throughout the body. Amyloid-associated diseases are challenging to study because amyloid formation occurs on time scales that span several orders of magnitude and involve heterogeneous, interconverting protein conformations. The development of more effective technologies to diagnose and treat amyloid disease requires both a map of the conformations sampled during amyloidogenesis and an understanding of the molecular mechanisms that drive this process. In prior molecular dynamics simulations of amyloid proteins, we observed the formation of a nonstandard type of secondary structure, called α-sheet, that we proposed is associated with the pathogenic conformers in amyloid disease, the soluble oligomers. However, the detailed molecular interactions that drive the conversion to α-sheet remain elusive. Here we use molecular dynamics simulations to interrogate a critical event in transthyretin aggregation, the formation of aggregation-competent, monomeric species. We show that conformational changes in one of the two ß-sheets in transthyretin enable solvent molecules and polar side chains to form electrostatic interactions with main-chain peptide groups to facilitate and modulate conversion to α-sheet secondary structure. Our results shed light on the early conformational changes that drive transthyretin toward the α-sheet structure associated with toxicity. Delineation of the molecular events that lead to aggregation at atomic resolution can aid strategies to target the early, critical toxic soluble oligomers.

Cardiac myosin activation with 2-deoxy-ATP via increased electrostatic interactions with actin.

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

α-Sheet secondary structure in amyloid ß-peptide drives aggregation and toxicity in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the deposition of ß-sheet-rich, insoluble amyloid ß-peptide (Aß) plaques; however, plaque burden is not correlated with cognitive impairment in AD patients; instead, it is correlated with the presence of toxic soluble oligomers. Here, we show, by a variety of different techniques, that these Aß oligomers adopt a nonstandard secondary structure, termed "α-sheet." These oligomers form in the lag phase of aggregation, when Aß-associated cytotoxicity peaks, en route to forming nontoxic ß-sheet fibrils. De novo-designed α-sheet peptides specifically and tightly bind the toxic oligomers over monomeric and fibrillar forms of Aß, leading to inhibition of aggregation in vitro and neurotoxicity in neuroblastoma cells. Based on this specific binding, a soluble oligomer-binding assay (SOBA) was developed as an indirect probe of α-sheet content. Combined SOBA and toxicity experiments demonstrate a strong correlation between α-sheet content and toxicity. The designed α-sheet peptides are also active in vivo where they inhibit Aß-induced paralysis in a transgenic Aß Caenorhabditis elegans model and specifically target and clear soluble, toxic oligomers in a transgenic APPsw mouse model. The α-sheet hypothesis has profound implications for further understanding the mechanism behind AD pathogenesis.

Visualizing Protein Folding and Unfolding.

Protein folding/unfolding is a complicated process that defies high-resolution characterization by experimental methods. As an alternative, atomistic molecular dynamics simulations are now routinely employed to elucidate and magnify the accompanying conformational changes and the role of solvent in the folding process. However, the level of detail necessary to map the process at high spatial-temporal resolution provides an overwhelming amount of data. As more and better tools are developed for analysis of these large data sets and validation of the simulations, one is still left with the problem of visualizing the results in ways that provide insight into the folding/unfolding process. While viewing and interrogating static crystal structures has become commonplace, more and different approaches are required for dynamic, interconverting, unfolding, and refolding proteins. Here we review a variety of approaches, ranging from straightforward to complex and unintuitive for multiscale analysis and visualization of protein folding and unfolding.

The Role of α-sheet in Amyloid Oligomer Aggregation and Toxicity.

A major barrier to developing effective treatments and diagnostics for amyloid diseases is the inability of traditional protein structure characterization methods to elucidate the structure of the toxic oligomers that form during amyloidogenesis. Some years ago, our lab "discovered" a novel protein secondary structure in molecular dynamics simulations of multiple unrelated amyloid proteins, which we call α-sheet. We hypothesize that α-sheet plays an important role in amyloid aggregation and oligomer toxicity. De novo monomeric α-sheet peptides designed to be complementary to the structure observed in simulations inhibit amyloid aggregation and toxicity and specifically bind to the toxic oligomeric species in a variety of unrelated mammalian and bacterial amyloid systems associated with a range of diseases. Furthermore, spectroscopic analysis of α-sheet structure, including nuclear magnetic resonance (NMR), circular dichroism (CD), and Fourier-transform infrared spectroscopy (FTIR), correspond well to values predicted for α-sheet. These α-sheet designs are now being tested for their ability to detect and neutralize toxic oligomers in animals and in patient samples, demonstrating the potential of this nonstandard secondary structure as a target for therapeutic and diagnostic agents for amyloid diseases.

A Carboxylate to Amide Substitution That Switches Protein Folds.

Metamorphic proteins are biomolecules prone to adopting alternative conformations. Because of this feature, they represent ideal systems to investigate the general rules allowing primary structure to dictate protein topology. A comparative molecular dynamics study was performed on the denatured states of two proteins, sharing nearly identical amino-acid sequences (88 %) but different topologies, namely an all-α-helical bundle protein named GA 88 and an α+ß-protein named GB 88. The analysis allowed successful design of and experimental validation of a site-directed mutant that promotes, at least in part, the switch in folding from GB 88 to GA 88. The mutated position, in which a glutamic acid was replaced by a glutamine, does not make any intramolecular interactions in the native state of GA 88, such that its stabilization can be explained by considering the effects on the denatured state. The results represent a direct demonstration of the role of the denatured state in sculpting native structure.

Molecular dynamics-derived rotamer libraries for d-amino acids within homochiral and heterochiral polypeptides.

Computational resources have contributed to the design and engineering of novel proteins by integrating genomic, structural and dynamic aspects of proteins. Non-canonical amino acids, such as d-amino acids, expand the available sequence space for designing and engineering proteins; however, the rotamer libraries for d-amino acids are usually constructed as the mirror images of l-amino acid rotamer libraries, an assumption that has not been tested. To this end, we have performed molecular dynamics (MD) simulations of model host-guest peptide systems containing d-amino acids. Our simulations systematically address the applicability of the mirror image convention as well as the effects of neighboring residue chirality. Rotamer libraries derived from these systems provide realistic rotamer distributions suitable for use in both rational and computational design workflows. Our simulations also address the impact of chirality on the intrinsic conformational preferences of amino acids, providing fundamental insights into the relationship between chirality and biomolecular dynamics. While d-amino acids are rare in naturally occurring proteins, they are used in designed proteins to stabilize a desired conformation, increase bioavailability or confer favorable biochemical and physical attributes. Here, we present d-amino acid rotamer libraries derived from MD simulations of alanine-based host-guest pentapeptides and show how certain residues can deviate from mirror image symmetry. Our simulations directly model d-amino acids as guest residues within the chiral l-Ala and d-Ala pentapeptide series to explicitly incorporate any contributions resulting from the chiralities of neighboring residues.