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Formation of protein knots is an intriguing offshoot of the protein folding problem. Since experimental resolution on knot formation is limited, theoretical methods currently provide the most detailed insights into the knotting process. While suitable for shallow knots, molecular dynamics simulations have faced challenges capturing the formation of deep knots in proteins such as the minimally tied trefoil α/β methyltransferase from

This review serves to highlight approaches that may improve the access of antibody drugs to regions of the brain affected by Alzheimer's Disease. While previous antibody drugs have been unsuccessful in treating Alzheimer's disease, recent work demonstrates that Alzheimer's pathology can be modified if these drugs can penetrate the brain parenchyma with greater efficacy. Research in antibody blood-brain barrier drug delivery predominantly follows one of three distinct directions: (1) enhancing influx with reduced antibody size, addition of Trojan horse modules, or blood-brain barrier disruption; (2) modulating trancytotic equilibrium and/or kinetics of the neonatal Fc Receptor; and (3) manipulation of antibody glycan carbohydrate composition. In addition to these topics, recent studies are discussed that reveal a role of glycan sialic acid in suppressing antibody efflux from the brain.

The data here consists of time-dependent experimental parameters from chemical and biophysical methods used to characterize Aβ monomeric reactants as well as soluble oligomer and amyloid fibril products from a slow (3–4 week) assembly reaction under biologically-relevant solvent conditions. The data of this reaction are both of a qualitative and quantitative nature, including gel images from chemical cross-linking and Western blots, fractional solubility, thioflavin T binding, size exclusion chromatograms, transmission electron microscopy images, circular dichroism spectra, and fluorescence resonance energy transfer efficiencies of donor–acceptor pair labels in the Aβ chain. This data enables future efforts to produce the initial monomer and eventual soluble oligomer and amyloid fibril states by providing reference benchmarks of these states pertaining to physical properties (solubility), ligand-binding (thioflavin T binding), mesoscopic structure (electron microscopy, size exclusion chromatography, cross-linking products, SDS and native gels) and molecular structure (circular dichroism, FRET donor-acceptor distance).Aβ1-40 soluble oligomers are produced that are suitable for biophysical studies requiring sufficient transient stability to exist in their “native” conformation in biological phosphate-saline buffers for extended periods of time. The production involves an initial preparation of highly monomeric Aβ in a phosphate saline buffer that transitions to fibrils and oligomers through time incubation alone, without added detergents or non-aqueous chemicals. This criteria ensures that the only difference between initial monomeric Aβ reactant and subsequent Aβ oligomer products is their degree of peptide assembly. A number of chemical and biophysical methods were used to characterize the monomeric reactants and soluble oligomer and amyloid fibril products, including chemical cross-linking, Western blots, fraction solubility, thioflvain T binding, size exclusion chromatography, transmission electron micrscopy, circular dichroism spectroscopy, and fluorescence resonance energy transfer.

Surface plasmon resonance was used to investigate the kinetics, affinity, and specificity of binding between anti-Aβ (beta-amyloid) IgG antibodies and oligomeric Aβ. Two factors were needed to accurately characterize the IgG binding kinetics. First, a bivalent model was necessary to properly fit the kinetic association and dissociation sensograms. Second, a high concentration of IgG was necessary to overcome a significant mass transport limitation that existed regardless of oligomer density on the sensor surface. Using high IgG concentrations and bivalent fits, consistent kinetic parameters were found at varying sensor surface ligand densities. A comparison of binding specificity, affinity, and kinetic flux between monoclonal and natural human anti-Aβ IgG antibodies revealed the following findings. First, monoclonal antibodies 6E10 and 4G8 single-site binding affinity is similar between Aβ oligomers and monomers. Second, natural human anti-Aβ IgG binding readily binds Aβ oligomers but does not bind monomers. Third, natural human anti-Aβ IgG binds Aβ oligomers with a higher affinity and kinetic flux than 6E10 and 4G8. Both the current analytical methodology and antibody binding profiles are important for advances in antibody drug development and kinetic biomarker applications for Alzheimer's disease.

Understanding protein tertiary and quaternary structures is crucial to a better understanding of their biological functions. Here we illustrate for tryptophan synthase that tandem mass spectrometry (MS/MS) reveals not only protein subunit architectures, but also protein unfolding behavior when coupled with ion mobility (IM). In the present study, we verified the subunit arrangement with surface induced dissociation (SID). We are able to correlate experimental results by IM with those obtained in unfolding simulations for the hetero-tetramer Tryptophan Synthase (TS) protein complex by identifying the presence of at least three stable intermediates (I1, I2, and I3) during the unfolding process in collision induced dissociation (CID). We illustrate that the unfolding of the TS complex is likely due to the initial unfolding of an α-monomer subunit, followed by the unfolding of the second α-monomers. We also illustrate the ability of this combination of techniques to not only identify conformational changes of TS upon addition of D,L-α-glycerol phosphate (GP), but also to determine the location of the ligand, which is buried within the α-monomer of the TS.

Background Drug delivery to the brain is a major roadblock to treatment of Alzheimer's disease. Recent results of the PRIME study indicate that increasing brain penetration of antibody drugs improves Alzheimer's treatment outcomes. New approaches are needed to better accomplish this goal. Based on prior evidence, the hypothesis that glycan modification alters antibody blood-brain barrier permeability was tested here. Methods The blood-brain barrier permeability coefficient Pe of different glycosylated states of anti-amyloid IgG was measured using in vitro models of brain microvascular endothelial cells. Monoclonal antibodies 4G8, with sialic acid, and 6E10, lacking sialic acid, were studied. The amount of sialic acid was determined using quantitative and semi-quantitative surface plasmon resonance methods. Results Influx of IgG was not saturable and was largely insensitive to IgG species and glycosylation state. By contrast, efflux of 4G8 efflux was significantly lower than both albumin controls and 6E10. Removal of α2,6-linked sialic acid group present on 12% of 4G8 completely restored efflux to that of 6E10 but increasing the α2,6-sialylated fraction to 15% resulted in no change. Removal of the Fc glycan from 4G8 partially restored efflux. Alternate sialic acid groups with α2,3 and α2,8 linkages, nor on the Fc glycan, were not detected at significant levels on either 4G8 or 6E10. Conclusions These results support a model in which surface-sialylated 4G8 inhibits its own efflux and that of asialylated 4G8. General significance Glycan modification has the potential to increase antibody drug penetration into the brain through efflux inhibition.

Polyglutamine repeats in proteins are highly correlated with amyloid formation and neurological disease. To better understand the molecular basis of glutamine repeat diseases, structural analysis of polyglutamine peptides as soluble monomers, oligomers, and insoluble amyloid fibrils is necessary. In this study, fluorescence resonance energy transfer (FRET) experiments and molecular dynamics simulations using different theoretical models of polyglutamine were conducted. This study demonstrates that a previously proposed simple CαCβ model of polyglutamine, denoted as FCO, accurately reproduced the present FRET results and the results of previously published FRET, triplet-state quenching, and fluorescence correlation studies. Other simple CαCβ models with random coil and extended β-strand parameters, and all-atom models with parm96 and parm99SB force fields, did not match the FRET result well. The FCO is an intrinsically disordered model with a high-effective persistence length producing extended peptides at short lengths (QN 16. Proteins 2011. © 2010 Wiley-Liss, Inc.

Sonogashira coupling of diacetyl 5-ethynyl-2′-deoxyuridine with diacetyl 5-iodo-2′-deoxyuridine gave the acylated ethynediyl-linked 2′-deoxyuridine dimer (3 b; 63 %), which was deprotected with ammonia/methanol to give ethynediyl-linked 2′-deoxyuridines (3 a; 79 %). Treatment of 5-ethynyl-2′-deoxyuridine (1 a) with 5-iodo-2′-deoxyuridine gave the furopyrimidine linked to 2′-deoxyuridine (78 %). Catalytic oxidative coupling of 1 a (O2, CuI, Pd/C, N,N-dimethylformamide) gave butadiynediyl-linked 2′-deoxyuridines (4; 84 %). Double Sonogashira coupling of 5-iodo-2′-deoxyuridine with 1,4-diethynylbenzene gave 1,4-phenylenediethynediyl-bridged 2′-deoxyuridines (5; 83 %). Cu-catalyzed cycloisomerization of dimers 4 and 5 gave their furopyrimidine derivatives. One-electron addition to 1 a, 3 a, and 4 gave the anion radical, the EPR spectra of which showed that the unpaired electron is largely localized at C6 of one uracil ring (17 G doublet) at 77 K. The EPR spectra of the one-electron-oxidized derivatives of ethynediyl- and butadiynediyl-linked uridines 3 a and 4 at 77 K showed that the unpaired electron is delocalized over both rings. Therefore, structures 3 a and 4 provide an efficient electronic link for hole conduction between the uracil rings. However, for the excess electron, an activation barrier prevents coupling to both rings. These dimeric structures could provide a gate that would separate hole transfer from electron transport between strands in DNA systems. In the crystal structure of acylated dimer 3 b, the bases were found in the anti position relative to each other across the ethynyl link, and similar anti conformation was preserved in the derived furopyrimidine–deoxyuridine dinucleoside.

The structures of partially folded states appearing during the folding of a (betaalpha)(8) TIM barrel protein, the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (betaalpha)(4) region, modest protection in the neighboring (betaalpha)(1-3) and (betaalpha)(5)beta(6) segments and no significant protection in the remaining N and C-terminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (betaalpha)(2-5)beta(6) region after 5 s of folding demonstrates that these species represent kinetically distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate, while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C(alpha) Gō model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of the structure offering protection against exchange in the on-pathway intermediate(s). Because the native-centric Gō model simulations do not explicitly include sequence-specific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō model simulation.

Kinetic simulations of the folding and unfolding of triosephosphate isomerase (TIM) from yeast were conducted using a single monomer gammaTIM polypeptide chain that folds as a monomer and two gammaTIM chains that fold to the native dimer structure. The basic protein model used was a minimalist Gō model using the native structure to determine attractive energies in the protein chain. For each simulation type--monomer unfolding, monomer refolding, dimer unfolding, and dimer refolding--thirty simulations were conducted, successfully capturing each reaction in full. Analysis of the simulations demonstrates four main conclusions. First, all four simulation types have a similar "folding order", i.e., they have similar structures in intermediate stages of folding between the unfolded and folded state. Second, despite this similarity, different intermediate stages are more or less populated in the four different simulations, with 1), no intermediates populated in monomer unfolding; 2), two intermediates populated with beta(2)-beta(4) and beta(1)-beta(5) regions folded in monomer refolding; 3), two intermediates populated with beta(2)-beta(3) and beta(2)-beta(4) regions folded in dimer unfolding; and 4), two intermediates populated with beta(1)-beta(5) and beta(1)-beta(5) + beta(6) + beta(7) + beta(8) regions folded in dimer refolding. Third, simulations demonstrate that dimer binding and unbinding can occur early in the folding process before complete monomer-chain folding. Fourth, excellent agreement is found between the simulations and MPAX (misincorporation proton alkyl exchange) experiments. In total, this agreement demonstrates that the computational Gō model is accurate for gammaTIM and that the energy landscape of gammaTIM appears funneled to the native state.

The role of native contact topology in the folding of a TIM barrel model based on the alpha-subunit of tryptophan synthase (alphaTS) from Salmonella typhimurium (Protein Data Bank structure 1BKS) was studied using both equilibrium and kinetic simulations. Equilibrium simulations of alphaTS reveal the population of two intermediate ensembles, I1 and I2, during unfolding/refolding at the folding temperature, Tf = 335 K. Equilibrium intermediate I1 demonstrates discrete structure in regions alpha0-beta6 whereas intermediate I2 is a loose ensemble of states with N-terminal structure varying from at least beta1-beta3 (denoted I2A) to alpha0-beta4 at most (denoted I2B). The structures of I1 and I2 match well with the two intermediate states detected in equilibrium folding experiments of Escherichia coli alphaTS. Kinetic folding simulations of alphaTS reveal the sequential population of four intermediate ensembles, I120Q, I200Q, I300Q, and I360Q, during refolding. Kinetic intermediates I120Q, I200Q, and I300Q are highly similar to equilibrium alphaTS intermediates I2A, I2B, and I1, respectively, consistent with kinetic experiments on alphaTS from E. coli. A small population (approximately 10%) of kinetic trajectories are trapped in the I120Q intermediate ensemble and require a slow and complete unfolding step to properly refold. Both the on-pathway and off-pathway I120Q intermediates show structure in beta1-beta3, which is also strikingly consistent with kinetic folding experiments of alphaTS. In the off-pathway intermediate I(120Q), helix alpha2 is wrapped in a nonnative chiral arrangement around strand beta3, sterically preventing the subsequent folding step between beta3 and beta4. These results demonstrate the success of combining kinetic and equilibrium simulations of minimalist protein models to explore TIM barrel folding and the folding of other large proteins.

A minimalist Go-model, with no energetic frustration in the native conformation, has been shown to describe accurately the folding pathway of the beta-trefoil protein, interleukin-1beta (IL-1beta). While it appears that these models successfully model transition states and intermediates between the unfolded and native ensembles, it is unclear how accurately they capture smaller, yet biologically relevant, structural changes within the native ensemble after energetic perturbation. Here, we address the following questions. Can a simple Go-model of interleukin-1beta, based on native topology, describe changes in structural properties of the native ensemble as the protein stability is changed? Or is it necessary to include a more explicit representation of atoms, electrostatic, hydrogen bonding, and van der Waals forces to describe these changes? The native ensemble of IL-1beta was characterized using a variety of experimental probes under native (0 M NaCl, guanidine hydrochloride (Gdn-HCl)), moderately destabilized (0 M NaCl, 0.8 M Gdn-HCl), and in moderate salt concentration (0.8 M NaCl, 0 M Gdn-HCl). Heteronuclear (1)H-(15)N nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) NMR spectra confirmed that the beta-trefoil global fold was largely intact under these three conditions. However, 25 of the 153 residues throughout the chain did demonstrate (13)C and (1)H-(15)N chemical shifts when perturbed with 0.8 M NaCl or Gdn-HCl. Despite large differences in protection factors from solvent hydrogen-deuterium exchange for all residues between stable (0 M Gdn-HCl) and destabilized (0.8 M Gdn-HCl) IL-1beta, no difference in steady-state (15)N-(1)H NOE enhancements were measured. Thus, the chemical shifts correlate with a global but limited increase in residue flexibility in the presence of Gdn-HCl. Minimalist simulations highlight the regions of greatest position shift between native and 0.8 M Gdn-HCl, which were determined experimentally. This correlation demonstrates that structural changes within the native ensemble of IL-1beta are, at least partially, governed by the principle of minimal energetic frustration.

models) have been successful in providing a qualitativeunderstanding of the folding mechanism of small globular proteins. Can we go beyond this qualitativeunderstanding and make more detailed quantitative connections to experiments? To achieve this goal, a tractableframework of protein representations whose complexity falls between C

Kinetic data measured from folding of the protein interleukin-1β fits best to three exponential phases when studied with tryptophan fluorescence but only two exponential phases when measured using other methods. The technique of ANS fluorescence was used to determine whether the additional phase observed in tryptophan fluorescence was also detected with ANS dye binding. Unlike trytophan fluorescence, the ANS fluorescence was highly dependent on the concentration of protein present during the folding experiment. Experimental controls provide evidence that ANS binds to protein aggregates, present at higher concentrations and absent at lower concentrations. Protein concentration-dependent folding studies demonstrate that, at lower interleukin-1β concentrations, tryptophan fluorescence kinetics can be fit adequately with a two exponential fit. This study indicates that (1) measured interleukin-1β folding kinetics fit to a 2 phase model and (2) at higher protein concentrations, transient association of IL-1β may result in a kinetic fit of 3 phases.

A point mutation, lysine 97 to isoleucine, in the all-beta cytokine interleukin-1 beta (IL-1 beta) exhibits an increased propensity to form inclusion bodies in vivo and aggregates in vitro. In an effort to better understand the aggregation reaction and determine when intervention may allow rescue of protein from aggregation during renaturation, we developed a novel application of mass spectrometry using isotopic labeling to determine the step(s) at which K97I commits to either the native or aggregated state. Interestingly, despite the early formation of a folding intermediate ensemble at an observed rate lambda(2) of 4.0 s(-1), K97I commits to folding at a significantly slower rate lambda(CF) of 0.021 s(-1). This rate of commitment to folding is in excellent agreement with the observed rate of K97I native state formation (lambda(1) = 0.018 s(-1)). K97I also commits slowly to aggregation at an observed rate lambda(CA) of 0.023 s(-1). Earlier folding species and aggregates present prior to these commitment steps are likely to be in a reversible equilibrium between monomeric folding intermediates and higher-order oligomers. Kinetic and equilibrium experimental measurements of folding and aggregation processes are consistent with a nucleation-dependent model of aggregation.

A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1beta (IL-1beta), exhibits increased levels of inclusion body (IB) formation relative to the wild-type protein (WT) when expressed in Escherichia coli. Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT. We examined the folding pathway of the mutant and wild-type proteins at pH 6.5 and 25 degrees C with manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated species in vitro correlate with the observation of increased aggregation in vivo. The refolding reactions of the WT and K97I proteins are both described by three exponential processes. Two exponential processes characterize fast events (0.1-1.0 s) in folding while the third exponential process correlates with a slow (70 s) single pathway to and from the native state. The K97I replacement affects the earlier steps in the refolding pathway. Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical protein concentration of 18 microM. This observation is consistent with an initial nucleation step mediating protein aggregation. Stopped-flow kinetic studies of the K97I aggregation process demonstrate that K97I aggregates most rapidly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is low. Folding and aggregation studies together support a model in which the formation of stable folding intermediates afford protection against further K97I aggregation.

Background The literature contains conflicting results regarding the status of serum anti-Aβ antibody concentrations in Alzheimer's disease (AD). Reduced levels of these antibodies have been suggested to contribute to the development of this disorder. The conflicting results may be due to polyvalent antibodies, antibody "masking" due to Aβ binding, methodological differences, and/or small sample sizes. The objectives of this pilot study were to compare serum anti-Aβ antibody concentrations between AD, mild cognitive impairment (MCI), and elderly noncognitively impaired (NCI) subjects while addressing these issues, and to perform power analyses to determine appropriate group sizes for future studies employing this approach. Methods Serum antibodies to Aβ1-42 monomer and soluble oligomers in AD, MCI, and NCI subjects (10/group) were measured by ELISA, subtracting polyvalent antibody binding and dissociating antibody-antigen complexes. Differences in mean antibody levels were assessed for significance with repeated measures ANOVA using restricted maximum likelihood estimation, using Tukey-Kramer tests and confidence intervals for multiple comparisons. Spearman's rank correlation was used to determine associations between anti-monomer and anti-oligomer antibody concentrations. Estimated sample sizes required to detect effects of various sizes were calculated. Results There were no significant differences between groups for mean anti-Aβ antibody levels, although these tended to be higher in AD than NCI specimens. Estimated group sizes of 328 and 150 for anti-Aβ monomer and oligomer antibodies, respectively, would have been required for 80% power for significance at 0.05 for a 25% increase in the AD mean relative to the NCI mean. Serum antibody concentrations to Aβ monomer and oligomers were strongly associated (correlations: 0.798 for undissociated sera, 0.564 for dissociated sera). Antibody-antigen dissociation significantly increased anti-Aβ monomer but not anti-Aβ oligomer antibody levels. Conclusions The findings in this pilot study are consistent with relatively similar concentrations of specific, non-antigen-bound antibodies to Aβ1-42 monomer and soluble oligomers in AD, MCI, and NCI sera. The differences between groups for these antibodies would have required approximate group sizes of 328 and 150, respectively, for a high probability for statistical significance. These findings do not support the hypothesis that reduced levels of anti-Aβ antibodies might contribute to AD's pathogenesis.

Plaques containing fibrillar amyloid-beta (Abeta) are a characteristic finding in Alzheimer's disease. Although plaque counts correlate poorly with the extent of cognitive deficits in this disorder, fibrillar Abeta can promote neuronal damage through a variety of mechanisms. External beam radiotherapy has been reported to be an effective treatment for tracheobronchial amyloidosis, in which amyloid is deposited as submucosal plaques and tumor-like masses in the trachea and/or bronchi. Radiotherapy's effectiveness in this disorder is thought to be due to its toxicity to plasma cells, but direct effects of radiotherapy on amyloid may also be involved. On this basis, whole-brain radiotherapy has been suggested as a treatment for Alzheimer's disease. The objective of this study was to determine the effects of external beam radiation on preformed Abeta1-42 fibrils and on the formation of these fibrils. Using the Thioflavin-T assay, no effects of radiation were found on either of these parameters. Our results in this in vitro study suggest that whole-brain irradiation is unlikely to directly reduce plaque counts in the Alzheimer's disease brain. This treatment might still lower plaque counts indirectly, but any potential benefits would need to be weighed against its possible neurotoxic effects, which could induce further cognitive deficits.

Polyglutamine repeats in proteins are highly correlated with amyloid formation and neurological disease. To better understand the molecular basis of glutamine repeat diseases, structural analysis of polyglutamine peptides as soluble monomers, oligomers, and insoluble amyloid fibrils is necessary. In this study, fluorescence resonance energy transfer (FRET) experiments and molecular dynamics simulations using different theoretical models of polyglutamine were conducted. This study demonstrates that a previously proposed simple C(α)C(β) model of polyglutamine, denoted as FCO, accurately reproduced the present FRET results and the results of previously published FRET, triplet-state quenching, and fluorescence correlation studies. Other simple C(α)C(β) models with random coil and extended β-strand parameters, and all-atom models with parm96 and parm99SB force fields, did not match the FRET result well. The FCO is an intrinsically disordered model with a high-effective persistence length producing extended peptides at short lengths (Q(N)10). Because of an increasing number of attractive Q-Q interactions at longer lengths, the FCO model becomes increasingly more compact at lengths between Q(N) ∼ 10-16 and is as compact as many folded proteins at Q(N)16.

Abeta soluble oligomers are believed to play a key role in the development of Alzheimer's disease (AD). An enzyme-linked immunosorbent assay (ELISA) commonly used to measure these proteins uses the same monoclonal antibody as both capture and reporter antibody. The objective of this study was to examine the specificity and sensitivity of this procedure, using monoclonal anti-Abeta antibody 6E10 as capture antibody and biotinylated 6E10 as reporter antibody. At comparable concentrations of Abeta soluble oligomers and low molecular weight (LMW) Abeta peptides, optical density (OD) values were four- to five-fold higher for the oligomer preparation than for the LMW Abeta. The LMW Abeta preparation, when evaluated by western blots of gels run under native conditions, showed only one band even after storage at 4 °C for more than two months, suggesting that the ELISA was detecting Abeta monomer as well as Abeta oligomers. Possible explanations for these results are that (1) the LMW Abeta preparation may contain Abeta oligomer species below the limit of detection of western blot, but still detectable by ELISA, or (2) some nonspecific binding of the LMW Abeta to the ELISA plate may have occurred, allowing its relevant epitope to remain available for binding by the reporter antibody. Because of the possibility that this ELISA may not be oligomer-specific, it seems prudent to suggest that it should be used in combination with other methods, rather than as the sole technique, for measuring Abeta oligomers in biological specimens.

A fluorescently labeled 20-residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E20) was used to study structural changes which occur in E20 as it co-aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o-aminobenzamide donor at the N-terminus and 3-nitrotyrosine acceptor at the C-terminus of E20. PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from α-helical monomers to aggregated β-sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time-resolved and steady-state RET measurements reveal four dominant E20 conformations: (1) a partially collapsed conformation (24 A donor–acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 A donor–acceptor distance), (3) a minor partially collapsed conformation (22 A donor-acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 A donor–acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom-level structural details of soluble oligomer and aggregated states of proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 299–317, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Improvement in cognitive scores in patients with Alzheimer's disease (AD) has been reported in two trials in which intravenous immunoglobulin (IvIg) preparations were administered. IvIg's benefits in AD patients have been suggested to be due to antibodies to amyloid-beta (Abeta). Our previous study using indirect enzyme-linked immunosorbent assay (ELISA) indicated that much of IvIg's apparent binding to Abeta1-42 is nonspecific; it is detectable even when IvIg is incubated on "specificity controls" (bovine serum albumin [BSA] and Abeta reverse sequence Abeta42-1) rather than Abeta1-42. The objective of this study was to evaluate procedures that might reduce this nonspecific binding. The IvIg preparation used was Gamunex (Talecris Biotherapeutics). Multiple blocking agents were evaluated, but even the most effective blocker only reduced nonspecific binding by 48%. Dissociating Gamunex's antibody-antigen complexes had no effect on specific binding when Abeta42-1 was used as the specificity control, although it increased this binding when BSA was the specificity control. Decreasing Gamunex's dilution from 1:1,500 to 1:500 resulted in a slight (7.4%) but significant (p=0.027) increase in specific binding. Using a sandwich ELISA to measure Gamunex's anti-Abeta antibodies resulted in even less specific binding to Abeta1-42 than with the indirect ELISA. Despite Gamunex's low percentage of specific binding to Abeta1-42, it inhibited Abeta oligomer formation. We conclude that, when anti-Abeta antibodies in IvIg are measured by indirect ELISA, extensive nonspecific binding occurs despite procedures taken to prevent it. This must be subtracted from total binding to accurately measure specific anti-Abeta antibody concentrations.

Cognitive improvement in Alzheimer's disease (AD) patients treated with intravenous immunoglobulin (IvIg) has been attributed to its antibodies to amyloid beta (Abeta). We compared the concentrations of specific antibodies to soluble Abeta1-42 conformations, namely Abeta1-42 monomer and Abeta1-42 soluble oligomers, between three IvIg preparations, Gamunex, Gammagard, and Flebogamma. To determine specific antibody concentrations to these Abeta1-42 conformations, nonspecific binding of the IvIg preparations to the Abeta reverse sequence, Abeta42-1, was subtracted. These antibodies were measured in untreated IvIg preparations and also after they were treated to dissociate antibody-antigen complexes, because this procedure has been reported to increase the detectable levels of serum anti-Abeta antibodies. Antibody levels to Abeta1-42 monomer were significantly higher in untreated Gamunex than in the other two IvIg preparations, and antibody-antigen dissociation increased the measured anti-Abeta monomer concentrations in Gamunex and Gammagard. Dissociated Gamunex and Gammagard had higher anti-Abeta monomer levels than Flebogamma. Generally similar results were found for antibodies to soluble Abeta1-42 oligomers, with the exception that after antibody-antigen dissociation, only Gammagard had significantly higher antibody levels than Flebogamma. These differences in antibody concentrations to Abeta1-42 conformations (particularly to Abeta1-42 soluble oligomers, thought to be the most neurotoxic conformation of soluble Abeta) and the increased availability of these antibodies after antibody-antigen complex dissociation have important implications for IvIg treatment of AD patients.

Fluorescence Resonance Energy Transfer (FRET) was used to study protein structures within soluble oligomer intermediates of amyloid and non-amyloid aggregates. Three protein aggregation systems were studied using fluorescence donor and acceptor near the N and C terminus:(1) Alzheimer's Aβ1-40 peptide.(2) 20-residue polyglutamine K2Q16K2.(3) 20-residue polyglutamic acid E20. (non-amyloid)The aggregation of each peptide showed different conformational changes: Aβ1-40 partially compacted into a structure consistent with solid-state NMR structures, K2Q16K2 extended to form β-sheets, and E20 compacted heavily into β-hairpins. However, based on donor-acceptor distances, soluble oligomers conformations of all three peptides show a remarkable degree of similarity to their monomeric precursors, albeit with a slightly expanded conformation for Aβ1-40 and E20. These findings support assembly models of small soluble oligomers which largely consist of monomer-like structures, with some increase in α-helical content for Aβ1-40. These donor-acceptor distances are used to directly assess the accuracy of different molecular dynamics force fields in the study of these soluble oligomers.

Recent experimental studies suggest that the mature GFP has an unconventional landscape composed of an early folding event with a typical funneled landscape, followed by a very slow search and rearrangement step into the locked, active chromophore-containing structure. As we have shown previously, the substantial difference in time scales is what generates the observed hysteresis in thermodynamic folding. The interconversion between locked and the soft folding structures at intermediate denaturant concentrations is so slow that it is not observed under the typical experimental observation time. Simulations of a coarse-grained model were used to describe the fast folding event as well as identify native-like intermediates on energy landscapes enroute to the fluorescent native fold. Interestingly, these simulations reveal structural features of the slow dynamic transition to chromophore activation. Experimental evidence presented here shows that the trapped, native-like intermediate has structural heterogeneity in residues previously linked to chromophore formation. We propose that the final step of GFP folding is a “locking” mechanism leading to chromophore formation and high stability. The combination of previous experimental work and current simulation work is explained in the context of a dual-basin folding mechanism described above.

Kinetic simulations of the folding and unfolding of the mammalian TIM barrel protein aldolase were conducted to determine if a minimalist monomeric Gō model, using the native structure to determine attractive energies in the protein chain, could capture the experimentally determined folding pathway. The folding order, that is, the order in which different secondary structures fold, between the Gō model simulations and that from hydrogen-deuterium exchange experiments, did not agree. To explain this discrepancy, two alternate variant of the basic Gō model were simulated: (1) a monomer Gō model with native contact energies weighted by a statistical potential (SP model) and (2) a monomer Gō model with native contact energies inversely weighted by crystallographic B factors (B model). The B model demonstrated the best agreement between simulation and experiments. The success of the B model is attributed to the ability of B factors to highlight local energetic frustration in the aldolase structure which results in weaker native contacts in these frustrated regions. The predictive success of the B model also reveals the potential use of B factor information for energetic weighting in general protein modeling.

The equilibrium structural ensemble of a 20-residue polyglutamic acid peptide (E(20)) was studied with FRET, circular dichroism, and molecular dynamics (MD) simulations. A FRET donor, o-aminobenzamide, and acceptor, 3-nitrotyrosine, were introduced at the N- and C-termini, respectively. Circular dichroism, steady state FRET, and time-resolved FRET measurements were employed to characterize the fraction helix and end-to-end distance under different pH conditions: pH 4 (60% alpha-helix), pH 6 (0% alpha-helix), and pH 9 (0% alpha-helix). At pH 4, the end-to-end distance was measured at 24 A and determined to be considerably less than the 31 A predicted for an alpha-helix of the same length. At pH 6 and 9, the end-to-end distance was measured at > 31 and 39 A respectively, both which are determined to be considerably greater than the 27 A predicted for a freely jointed random coil of the same length. To better understand the physical forces underlying the unusual helix-coil transition in this peptide, three theoretical MD models of E(20) were constructed: (1) a pure alpha-helix, (2) an alpha-helix with equivalent attractive intramolecular contacts, and (3) a weak alpha-helix with termini-weighted intramolecular contacts ("sticky ends"). Using MD simulations, the bent helix structure calculated from Model 3 was found to be the closest in agreement with the experimental data.

The fraction of IgG antibodies with anti-oligomeric Aβ affinity and surface sialic acid was compared between Octagam and Gammagard intravenous immunoglobulin (IVIG) using two complementary surface plasmon resonance methods. These comparisons were performed to identify if an elevated fraction existed in Gammagard, which reported small putative benefits in a recent Phase III clinical trial for Alzheimer’s Disease. The fraction of anti-oligomeric Aβ IgG was found to be higher in Octagam, for which no cognitive benefits were reported. The fraction and location of surface-accessible sialic acid in the Fab domain was found to be similar between Gammagard and Octagam. These findings indicate that anti-oligomeric Aβ IgG and total surface sialic acid alone cannot account for reported clinical differences in the two IVIG products. A combined analysis of sialic acid in anti-oligomeric Aβ IgG did reveal a notable finding that this subgroup exhibited a high degree of surface sialic acid lacking the conventional α2,6 linkage. These results demonstrate that the IVIG antibodies used to engage oligomeric Aβ in both Gammagard and Octagam clinical trials did not possess α2,6-linked surface sialic acid at the time of administration. Anti-oligomeric Aβ IgG with α2,6 linkages remains untested as an AD treatment.

The thermodynamic stability and folding kinetics of the all beta-sheet protein interleukin-1beta were measured between 0 and 4 M GdmCl concentrations and pH 5-7. Native interleukin-1beta undergoes a 3.5 kcal/mol decrease in thermodynamic stability, Delta, as pH is increased from 5 to 7. The native state parameter m(NU), measuring protein destabilization/[GdmCl], remains constant between pH 5 and 7, indicating that the solvent-exposed surface area difference between the native state and unfolded ensemble is unchanged across this pH range. Similarly, pH changes between 5 and 7 decrease only the thermodynamic stability, DeltaG(H)2(O), and not the m-values, of the kinetic intermediate and transition states. This finding is shown to be consistent with transition state configurations which continue to be the high-energy configurations of the transition state in the face of changing stability conditions. A three-state folding mechanism U right arrow over left arrow I right arrow over left arrow N is shown to be sufficient in characterizing IL-1beta folding under all conditions studied. The m-values of refolding transitions are much larger than the m-values of unfolding transitions, indicating that that the fast, T(2) (U right arrow over left arrow I), and slow, T(1) (I right arrow over left arrow N), transition states are highly similar to the intermediate I and native state N, respectively. Many of the folding properties of interleukin-1beta are shared among other members of the beta-trefoil protein family, although clear differences can exist.

Modeling the structure of natively disordered peptides has proved difficult due to the lack of structural information on these peptides. In this work, we use a novel application of the host-guest method, combining folding theory with experiments, to model the structure of natively disordered polyglutamine peptides. Initially, a minimalist molecular model (C(alpha)C(beta)) of CI2 is developed with a structurally based potential and captures many of the folding properties of CI2 determined from experiments. Next, polyglutamine "guest" inserts of increasing length are introduced into the CI2 "host" model and the polyglutamine is modeled to match the resultant change in CI2 thermodynamic stability between simulations and experiments. The polyglutamine model that best mimics the experimental changes in CI2 thermodynamic stability has 1), a beta-strand dihedral preference and 2), an attractive energy between polyglutamine atoms 0.75-times the attractive energy between the CI2 host Go-contacts. When free-energy differences in the CI2 host-guest system are correctly modeled at varying lengths of polyglutamine guest inserts, the kinetic folding rates and structural perturbation of these CI2 insert mutants are also correctly captured in simulations without any additional parameter adjustment. In agreement with experiments, the residues showing structural perturbation are located in the immediate vicinity of the loop insert. The simulated polyglutamine loop insert predominantly adopts extended random coil conformations, a structural model consistent with low resolution experimental methods. The agreement between simulation and experimental CI2 folding rates, CI2 structural perturbation, and polyglutamine insert structure show that this host-guest method can select a physically realistic model for inserted polyglutamine. If other amyloid peptides can be inserted into stable protein hosts and the stabilities of these host-guest mutants determined, this novel host-guest method may prove useful to determine structural preferences of these intractable but biologically relevant protein fragments.