Role of connecting loop I in catalysis and allosteric regulation of human glucokinase

Glucokinase (GCK, hexokinase IV) is a monomeric enzyme with a single glucose binding site that displays steady‐state kinetic cooperativity, a functional characteristic that affords allosteric regulation of GCK activity. Structural evidence suggests that connecting loop I, comprised of residues 47–71, facilitates cooperativity by dictating the rate and scope of motions between the large and small domains of GCK. Here we investigate the impact of varying the length and amino acid sequence of connecting loop I upon GCK cooperativity. We find that sequential, single amino acid deletions from the C‐terminus of connecting loop I cause systematic decreases in cooperativity. Deleting up to two loop residues leaves the k_cat value unchanged; however, removing three or more residues reduces k_cat by 1000‐fold. In contrast, the glucose K_0.5 and K_D values are unaffected by shortening the connecting loop by up to six residues. Substituting alanine or glycine for proline‐66, which adopts a cis conformation in some GCK crystal structures, does not alter cooperativity, indicating that cis/trans isomerization of this loop residue does not govern slow conformational reorganizations linked to hysteresis. Replacing connecting loop I with the corresponding loop sequence from the catalytic domain of the noncooperative isozyme human hexokinase I (HK‐I) eliminates cooperativity without impacting the k_cat and glucose K_0.5 values. Our results indicate that catalytic turnover requires a minimal length of connecting loop I, whereas the loop has little impact upon the binding affinity of GCK for glucose. We propose a model in which the primary structure of connecting loop I affects cooperativity by influencing conformational dynamics, without altering the equilibrium distribution of GCK conformations.     

First evidence of changes in enzyme kinetics and stability of glucokinase affected by somatic cancer-associated variations

Recent investigation of somatic variations of allosterically regulated proteins in cancer genomes suggested that variations in glucokinase (GCK) might play a role in tumorigenesis. We hypothesized that somatic cancer-associated GCK variations include in part those with activating and/or stabilizing effects. We analyzed the enzyme kinetics and thermostability of recombinant proteins possessing the likely activating variations and the variations present in the connecting loop I and provided the first experimental evidence of the effects of somatic cancer-associated GCK variations. Activating and/or stabilizing variations were common among the analyzed cancer-associated variations, which was in strong contrast to their low frequency among germinal variations. The activating and stabilizing variations displayed focal distribution with respect to the tertiary structure, and were present in the surroundings of the heterotropic allosteric activator site, including but not limited to the connecting loop I and in the active site region subject to extensive rearrangements upon glucose binding. Activating somatic cancer-associated variations induced a reduction of GCK's cooperativity and an increase in the affinity to glucose (a decline in the S_0.5 values). The hotspot-associated variations, which decreased cooperativity, also increased the half-maximal inhibitory concentrations of the competitive GCK inhibitor, N-acetylglucosamine. Concluded, we have provided the first convincing biochemical evidence establishing GCK as a previously unrecognized enzyme that contributes to the reprogramming of energy metabolism in cancer cells. Activating GCK variations substantially increase affinity of GCK to glucose, disrupt the otherwise characteristic sigmoidal response to glucose and/or prolong the enzyme half-life. This, combined, facilitates glucose phosphorylation, thus supporting glycolysis and associated pathways.     

Order–Disorder Transitions Govern Kinetic Cooperativity and Allostery of Monomeric Human Glucokinase

Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body''s principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity–onset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK''s homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder–order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder–order cycle of the small domain as a “time-delay loop,” which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.     

Regulation of Glucokinase by Intracellular Calcium Levels in Pancreatic β Cells

Glucokinase (GCK) controls the rate of glucose metabolism in pancreatic β cells, and its activity is rate-limiting for insulin secretion. Posttranslational GCK activation can be stimulated through either G protein-coupled receptors or receptor tyrosine kinase signaling pathways, suggesting a common mechanism. Here we show that inhibiting Ca²⁺ release from the endoplasmic reticulum (ER) decouples GCK activation from receptor stimulation. Furthermore, pharmacological release of ER Ca²⁺ stimulates activation of a GCK optical biosensor and potentiates glucose metabolism, implicating rises in cytoplasmic Ca²⁺ as a critical regulatory mechanism. To explore the potential for glucose-stimulated GCK activation, the GCK biosensor was optimized using circularly permuted mCerulean3 proteins. This new sensor sensitively reports activation in response to insulin, glucagon-like peptide 1, and agents that raise cAMP levels. Transient, glucose-stimulated GCK activation was observed in βTC3 and MIN6 cells. An ER-localized channelrhodopsin was used to manipulate the cytoplasmic Ca²⁺ concentration in cells expressing the optimized FRET-GCK sensor. This permitted quantification of the relationship between cytoplasmic Ca²⁺ concentrations and GCK activation. Half-maximal activation of the FRET-GCK sensor was estimated to occur at ∼400 nm Ca²⁺. When expressed in islets, fluctuations in GCK activation were observed in response to glucose, and we estimated that posttranslational activation of GCK enhances glucose metabolism by ∼35%. These results suggest a mechanism for integrative control over GCK activation and, therefore, glucose metabolism and insulin secretion through regulation of cytoplasmic Ca²⁺ levels.     

Hydration dependent dynamics in RNA

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms–ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in ²H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state ¹³C relaxation measurements, we establish that ns–μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.     

Insight into the biochemical characteristics of a novel glucokinase gene mutation

Glucokinase (GCK) acts as a glucose sensor and regulates β-cell insulin secretion. The heterozygous mutations in the gene encoding GCK cause a reduction of the enzyme activity, which results in a monogenic form of diabetes, maturity-onset diabetes of the young. In the present study, we identified and functionally characterized a novel missense mutation in the GCK gene, which results in a protein mutation Glu³³⁹ → Lys (E339K), from a Chinese family with hyperglycemia. The same GCK mutation that co-segregated with diabetes phenotype was identified in five members of this family but was not found in 200 healthy control individuals. We expressed and affinity-purified the GCK proteins from bacterial expression system that carries mutation (E339K) and fused to glutathione S-transferase. The expressed GCK protein was subjected to the measurement of its biochemical effects of the missense mutation on GCK activity. Our results showed that the mutation reduced the GCK protein yield. The enzymatic kinetics and the thermal stability analysis on the recombinant GCK proteins revealed that the mutation inactivates enzyme kinetics and severely impaired the GCK protein stability.     

Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease-Associated “Toggle” Calcium-Sensing Receptor Mutations

The Ca²⁺-sensing receptor (CaSR) regulates Ca²⁺ homeostasis in the body by monitoring extracellular levels of Ca²⁺ ([Ca²⁺]_o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca²⁺]_o–induced [Ca²⁺]_i oscillations, inositol-1-phosphate (IP₁) accumulation and extracellular signal-regulated kinases (ERK_1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca²⁺]_o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-of-function CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC₅₀ similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca²⁺]_o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu¹⁷³, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro²²¹ and Leu¹⁷³ are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding.     

How does it really move? Recent progress in the investigation of protein nanosecond dynamics by NMR and simulation

Nuclear magnetic resonance (NMR) spin relaxation experiments currently probe molecular motions on timescales from picoseconds to nanoseconds. The detailed interpretation of these motions in atomic detail benefits from complementarity with the results from molecular dynamics (MD) simulations. In this mini-review, we describe the recent developments in experimental techniques to study the backbone dynamics from ¹⁵N relaxation and side-chain dynamics from ¹³C relaxation, discuss the different analysis approaches from model-free to dynamics detectors, and highlight the many ways that NMR relaxation experiments and MD simulations can be used together to improve the interpretation and gain insights into protein dynamics.     

Chronic Ethanol Consumption-induced Pancreatic β-Cell Dysfunction and Apoptosis through Glucokinase Nitration and Its Down-regulation

Chronic ethanol consumption is known as an independent risk factor for type 2 diabetes, which is characterized by impaired glucose homeostasis and insulin resistance; however, there is a great deal of controversy concerning the relationships between alcohol consumption and the development of type 2 diabetes. We investigated the effects of chronic ethanol consumption on pancreatic β-cell dysfunction and whether generated peroxynitrite participates in the impaired glucose homeostasis. Here we show that chronic ethanol feeding decreases the ability of pancreatic β-cells to mediate insulin secretion and ATP production in coordination with the decrease of glucokinase, Glut2, and insulin expression. Specific blockade of ATF3 using siRNA or C-terminally deleted ATF3(ΔC) attenuated ethanol-induced pancreatic β-cell apoptosis or dysfunction and restored the down-regulation of glucokinase (GCK), insulin, and pancreatic duodenal homeobox-1 induced by ethanol. GCK inactivation and down-regulation were predominantly mediated by ethanol metabolism-generated peroxynitrite, which were suppressed by the peroxynitrite scavengers N^γ-monomethyl-l-arginine, uric acid, and deferoxamine but not by the S-nitrosylation inhibitor DTT, indicating that tyrosine nitration is the predominant modification associated with GCK down-regulation and inactivation rather than S-nitrosylation of cysteine. Tyrosine nitration of GCK prevented its association with pBad, and GCK translocation into the mitochondria results in subsequent proteasomal degradation of GCK following ubiquitination. This study identified a novel and efficient pathway by which chronic ethanol consumption may induce GCK down-regulation and inactivation by inducing tyrosine nitration of GCK, resulting in pancreatic β-cell apoptosis and dysfunction. Peroxynitrite-induced ATF3 may also serve as a potent upstream regulator of GCK down-regulation and β-cell apoptosis.     

Differential expression of genes and changes in glucose metabolism in the liver of liver-specific glucokinase gene knockout mice

To investigate the role of liver-specific expression of glucokinase (GCK) in the pathogenesis of hyperglycemia and to identify candidate genes involved in mechanisms of the onset and progression of maturity onset diabetes of the young, type 2 (MODY-2), we examined changes in biochemical parameters and gene expression in GCK knockout (gck^w/–) and wild-type (gck^w/w) mice as they aged. Fasting blood glucose levels were found to be significantly higher in the gck^w/– mice, compared to age-matched gck^w/w mice, at all ages (P < 0.05), except at 2 weeks. GCK activity of gck^w/– mice was about 50% of that of wild type (gck^w/w) mice (P < 0.05). Glycogen content at 4 and 40 weeks of age was lower in gck^w/– mice compared to gck^w/w mice. Differentially expressed genes in the livers of 2 and 26 week-old liver-specific GCK knockout (gck^w/–) mice were identified by suppression subtractive hybridization (SSH), which resulted in the identification of phosphoenolpyruvatecarboxykinase (PEPCK, also called PCK1) and Sterol O-acyltransferase 2 (SOAT2) as candidate genes involved in pathogenesis. The expressions of PEPCK and SOAT2 along with glycogen phosphorylase (GP) and glycogen synthase (GS) were then examined in GCK knockout (gck^w/–) and wild-type (gck^w/w) mice at different ages. Changes in PEPCK mRNA levels were confirmed by real-time RT-PCR, while no differences in the levels of expression of SOAT2 or GS were observed in age-matched GCK knockout (gck^w/–) and wild-type (gck^w/w) mice. GP mRNA levels were decreased in 40-week old gck^w/– mice compared to age-matched gck^w/w mice. Changes in gluconeogenesis, delayed development of GCK and impaired hepatic glycogen synthesis in the liver potentially lead to the onset and progression of MODY2.     

Conformational Dynamics and Cooperativity Drive the Specificity of a Protein-Ligand Interaction

Molecular recognition is critical for the fidelity of signal transduction in biology. Conversely, the disruption of protein-protein interactions can lead to disease. Thus, comprehension of the molecular determinants of specificity is essential for understanding normal biological signaling processes and for the development of precise therapeutics. Although high-resolution structures have provided atomic details of molecular interactions, much less is known about the influence of cooperativity and conformational dynamics. Here, we used the Tiam2 PSD-95/Dlg/ZO-1 (PDZ) domain and a quadruple mutant (QM), engineered by swapping the identity of four residues important for specificity in the Tiam1 PDZ into the Tiam2 PDZ domain, as a model system to investigate the role of cooperativity and dynamics in PDZ ligand specificity. Surprisingly, equilibrium binding experiments found that the ligand specificity of the Tiam2 QM was switched to that of the Tiam1 PDZ. NMR-based studies indicated that Tiam2 QM PDZ, but not other mutants, had extensive microsecond to millisecond motions distributed throughout the entire domain suggesting structural cooperativity between the mutated residues. Thermodynamic analyses revealed energetic cooperativity between residues in distinct specificity subpockets that was dependent upon the identity of the ligand, indicating a context-dependent binding mechanism. Finally, isothermal titration calorimetry experiments showed distinct entropic signatures along the mutational trajectory from the Tiam2 wild-type to the QM PDZ domain. Collectively, our studies provide unique insights into how structure, conformational dynamics, and thermodynamics combine to modulate ligand-binding specificity and have implications for the evolution, regulation, and design of protein-ligand interactions.     

Preparation, resonance assignment, and preliminary dynamics characterization of residue specific 13C/15N-labeled elongated DNA for the study of sequence-directed dynamics by NMR

DNA is a highly flexible molecule that undergoes functionally important structural transitions in response to external cellular stimuli. Atomic level spin relaxation NMR studies of DNA dynamics have been limited to short duplexes in which sensitivity to biologically relevant fluctuations occurring at nanosecond timescales is often inadequate. Here, we introduce a method for preparing residue-specific ¹³C/¹⁵N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply the approach to probe fast inter-helical bending motions induced by an adenine tract. Preliminary results suggest the presence of elevated A-tract independent end-fraying internal motions occurring at nanosecond timescales, which evade detection in short DNA constructs and that penetrate deep (7 bp) within the DNA helix and gradually fade away towards the helix interior.     

Association with nitric oxide synthase on insulin secretory granules regulates glucokinase protein levels

Glucokinase (GCK) association with insulin-secretory granules is controlled by interaction with nitric oxide synthase (NOS) and is reversed by GCK S-nitrosylation. Nonetheless, the function of GCK sequestration on secretory granules is unknown. Here we report that the S-nitrosylation blocking V367M mutation prevents GCK accumulation on secretory granules by inhibiting association with NOS. Expression of this mutant is reduced compared with a second S-nitrosylation blocking GCK mutant (C371S) that accumulates to secretory granules and is expressed at levels greater than wild type. Even so, the rate of degradation for wild type and mutant GCK proteins were not significantly different from one another, and neither mutation disrupted the ability of GCK to be ubiquitinated. Furthermore, gene silencing of NOS reduced endogenous GCK content but did not affect β-actin content. Treatment of GCK(C371S) expressing cells with short interfering RNA specific for NOS also blocked accumulation of this protein to secretory granules and reduced expression levels to that of GCK(V367M). Conversely, cotransfection of catalytically inactive NOS increased GCK-mCherry levels. Expression of GCK(C371S) in βTC3 cells enhanced glucose metabolism compared with untransfected cells and cells expressing wild type GCK, even though this mutant has slightly reduced enzymatic activity in vitro. Finally, molecular dynamics simulations revealed that V367M induces conformational changes in GCK that are similar to S-nitrosylated GCK, thereby suggesting a mechanism for V367M-inhibition of NOS association. Our findings suggest that sequestration of GCK on secretory granules regulates cellular GCK protein content, and thus cellular GCK activity, by acting as a storage pool for GCK proteins.     

Solid-state NMR approaches to measure topological equilibria and dynamics of membrane polypeptides

Biological membranes are characterized by a high degree of dynamics. In order to understand the function of membrane proteins and even more of membrane-associated peptides, these motional aspects have to be taken into consideration. Solid-state NMR spectroscopy is a method of choice when characterizing topological equilibria, molecular motions, lateral and rotational diffusion as well as dynamic oligomerization equilibria within fluid phase lipid bilayers. Here we show and review examples where the ¹⁵N chemical shift anisotropy, dipolar interactions and the deuterium quadrupolar splittings have been used to analyze motions of peptides such as peptaibols, antimicrobial sequences, Vpu, phospholamban or other channel domains. In particular, simulations of ¹⁵N and ²H-solid-state NMR spectra are shown of helical domains in uniaxially oriented membranes when rotation around the membrane normal or the helix long axis occurs.     

Elastic network models capture the motions apparent within ensembles of RNA structures

The role of structure and dynamics in mechanisms for RNA becomes increasingly important. Computational approaches using simple dynamics models have been successful at predicting the motions of proteins and are often applied to ribonucleo-protein complexes but have not been thoroughly tested for well-packed nucleic acid structures. In order to characterize a true set of motions, we investigate the apparent motions from 16 ensembles of experimentally determined RNA structures. These indicate a relatively limited set of motions that are captured by a small set of principal components (PCs). These limited motions closely resemble the motions computed from low frequency normal modes from elastic network models (ENMs), either at atomic or coarse-grained resolution. Various ENM model types, parameters, and structure representations are tested here against the experimental RNA structural ensembles, exposing differences between models for proteins and for folded RNAs. Differences in performance are seen, depending on the structure alignment algorithm used to generate PCs, modulating the apparent utility of ENMs but not significantly impacting their ability to generate functional motions. The loss of dynamical information upon coarse-graining is somewhat larger for RNAs than for globular proteins, indicating, perhaps, the lower cooperativity of the less densely packed RNA. However, the RNA structures show less sensitivity to the elastic network model parameters than do proteins. These findings further demonstrate the utility of ENMs and the appropriateness of their application to well-packed RNA-only structures, justifying their use for studying the dynamics of ribonucleo-proteins, such as the ribosome and regulatory RNAs.     

Multi-timescale dynamics study of FKBP12 along the rapamycin-mTOR binding coordinate

Drugs can affect function in proteins by modulating their flexibility. Despite this possibility, there are very few studies on how drug binding affects the dynamics of target macromolecules. FKBP12 (FK506 binding protein 12) is a prolyl cis-trans isomerase and a drug target. The immunosuppressant drug rapamycin exerts its therapeutic effect by serving as an adaptor molecule between FKBP12 and the cell proliferation regulator mTOR (mammalian target of rapamycin). To understand the role of dynamics in rapamycin-based immunosuppression and to gain insight into the role of dynamics in the assembly of supramolecular complexes, we used ¹⁵N, ¹³C, and ²H NMR spin relaxation to characterize FKBP12 along the binding coordinate that leads to cell cycle arrest. We show that sequential addition of rapamycin and mTOR leads to incremental rigidification of the FKBP12 backbone on the picosecond-nanosecond timescale. Both binding events lead to perturbation of main-chain and side-chain dynamics at sites distal to the binding interfaces, suggesting tight coupling interactions dispersed throughout the FKBP12-rapamycin interface. Binding of the first molecule, rapamycin, quenches microsecond-millisecond motions of the FKBP12 80's loop. This loop provides much of the surface buried at the protein-protein interface of the ternary complex, leading us to assert that preorganization upon rapamycin binding facilitates binding of the second molecule, mTOR. Widespread microsecond-millisecond motions of the backbone persist in the drug-bound enzyme, and we provide evidence that these slow motions represent coupled dynamics of the enzyme and isomerization of the bound drug. Finally, the pattern of microsecond-millisecond dynamics reported here in the rapamycin complex is dramatically different from the pattern in the complex with the structurally related drug FK506. This raises the important question of how two complexes that are highly isomorphic based on high-resolution static models have such different flexibilities in solution.     

Analysis of Sub-τc and Supra-τc Motions in Protein Gβ1 Using Molecular Dynamics Simulations

The functions of proteins depend on the dynamical behavior of their native states on a wide range of timescales. To investigate these dynamics in the case of the small protein Gβ1, we analyzed molecular dynamics simulations with the model-free approach of nuclear magnetic relaxation. We found amplitudes of fast timescale motions (sub-τ_c, where τ_c is the rotational correlation time) consistent with S² obtained from spin relaxation measurements as well as amplitudes of slow timescale motions (supra-τ_c) in quantitative agreement with S² order parameters derived from residual dipolar coupling measurements. The slow timescale motions are associated with the large variations of the ³J couplings that follow transitions between different conformational substates. These results provide further characterization of the large structural fluctuations in the native states of proteins that occur on timescales longer than the rotational correlation time.     

Dual effects of familial Alzheimer's disease mutations (D7H,D7N,and H6R) on amyloid β peptide: Correlation dynamics and zinc binding

Although the N‐terminal region of Amyloid β (Aβ) peptides plays dual roles as metal‐coordinating sites and conformational modulator, few studies have been performed to explore the effects of mutations at this region on the overall conformational ensemble of Aβ and the binding propensity of metal ions. In this work, we focus on how three familial Alzheimer's disease mutations (D7H, D7N, and H6R) alter the structural characteristics and thermodynamic stabilities of Aβ42 using molecular dynamics simulations. We observe that each mutation displays increased β‐sheet structures in both N and C termini. In particular, both the N terminus and central hydrophobic region of D7H can form stable β‐hairpin structures with its C terminus. The conserved turn structure at Val²⁴–Lys²⁸ in all peptides and Zn²⁺‐bound Aβ42 is confirmed as the common structural motif to nucleate folding of Aβ. Each mutant can significantly increase the solvation free energy and thus enhance the aggregation of Aβ monomers. The correlation dynamics between Aβ(1–16) and Aβ(17–42) fragments are elucidated by linking the domain motions with the corresponding structured conformations. We characterize the different populations of correlated domain motions for each mutant from a more macroscopic perspective, and unexpectedly find that Zn²⁺‐bound Aβ42 ensemble shares the same populations as Aβ42, indicating that the binding of Zn²⁺ to Aβ follows the conformational selection mechanism, and thus is independent of domain motions, even though the structures of Aβ have been modified at a residue level. Proteins 2014; 82:3286–3297. © 2014 Wiley Periodicals, Inc.