Biophysical Chemistry

Aggregation of intrinsically disordered amyloid {beta} (A{beta}) is a hallmark of Alzheimers disease. Although complex aggregation mechanisms have been increasingly revealed, structural ensembles of A{beta} monomers with heterogeneous and transient properties still hamper detailed experimental accesses to early events of amyloidogenesis. We herein developed a new mathematical tool based on multiple linear regression to obtain the reasonable ensemble structures of A{beta} monomer by using the solution nuclear magnetic resonance (NMR) and molecular dynamics simulation data. Our approach provided the best-fit ensemble to two-dimensional NMR chemical shifts, also consistent with circular dichroism and dynamic light scattering analyses. The major monomeric structures of A{beta} including {beta}-sheets in both terminal and central hydrophobic core regions and the minor partially-helical structures suggested initial structure-based explanation on possible mechanisms of early molecular association and nucleation for amyloid generation. A wide-spectrum application of the current approach was also indicated by showing a successful utilization for ensemble structures of folded proteins. We propose that multiple linear regression in combination to experimental results will be highly promising for studies on protein misfolding diseases and functions by providing a convincing template structure. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/457317v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1b4e6eeorg.highwire.dtl.DTLVardef@1a533eeorg.highwire.dtl.DTLVardef@f48eb3org.highwire.dtl.DTLVardef@1c4bf9a_HPS_FORMAT_FIGEXP M_FIG C_FIG

GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size [~]7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

We compared the conformations of the transmembrane domain (TMD) of influenza A M2 (IAM2) protein reconstituted at pH 7.4 in DOPC/DOPS bilayers to those in isolated E. coli membranes, having preserved its native proteins and lipids. IAM2 is a single-pass transmembrane protein known to assemble into homo-tetrameric proton channel. To represent this channel, we made a construct containing the IAM2s TMD region flanked by the juxtamembrane residues. The single cysteine substitute, L43C, of leucine located in the bilayer polar region was paramagnetically tagged with a methanethiosulfonate nitroxide label for the ESR (electron spin resonance) study. We compared the conformations of the spin-labeled IAM2 residing in DOPC/DOPS and native E. coli membranes using continuous-wave (CW) ESR and double electron-electron resonance (DEER) spectroscopy. The total protein-to-lipid molar ratio spanned the range from 1:230 to 1:10,400. The CW ESR spectra corresponded to a nearly rigid limit spin label dynamics in both environments. In all cases, the DEER data were reconstructed into the distance distributions showing well-resolved peaks at 1.68 nm and 2.37 nm. The peak distance ratio was 1.41{+/-}0.2 and the amplitude ratio was 2:1. This is what one expects from four nitroxide spin-labels located at the corners of a square, indicative of an axially symmetric tetramer. Distance modeling of DEER data with molecular modeling software applied to the NMR molecular structures (PDB: 2L0J) confirmed the symmetry and closed state of the C-terminal exit pore of the IAM2 tetramer in agreement with the NMR model. Thus, we can conclude that IAM2 TMD has similar conformations in model and native E. coli membranes of comparable thickness and fluidity, notwithstanding the complexity of the E. coli membranes caused by their lipid diversity and the abundance of integral and peripheral membrane proteins.

Gram-negative bacteria, such as E. coli and Salmonella, contain proteinaceous, hair-like, cell surface filaments known as curli. Curli serve to facilitate cell-cell interactions and are essential for host cell colonization. Curli assembly involves six proteins, CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. CsgE and CsgF are thought to act as chaperones to help prevent the premature aggregation of CsgA and/or CsgB, and to help transport these proteins, through the outer-membrane protein CsgG, to the cell surface where they assemble to form Curli. It has been observed that CsgF is able to inhibit the aggregation of CsgA, the major protein component of Curli. This article describes CsgFs ability to influence the aggregation of human islet amyloid polypeptide (hIAPP), an amyloidogenic polypeptide that is unrelated to Curli. In the presence of CsgF no increase in Thioflavin T fluorescence was observed for freshly solubilized hIAPP monitored as a function of time, suggesting that CsgF prevents the aggregation of hIAPP during the time period of observation. An analog of CsgF lacking the N-terminal unstructured region retained the ability to inhibit the aggregation of hIAPP. The nature of the CsgF-hIAPP interaction was probed via fluorescence quenching using a series of single cysteine mutants of CsgF labeled via the individual cysteine side chains with the fluorophore IAEDANS. In the presence of hIAPP, but not in the presence of the non-amyloidogenic rat islet amyloid polypeptide, the fluorophore attached to position of 23 of CsgF was found to be less exposed the quencher acrylamide suggesting that the interaction of hIAPP changes the solvent exposure of the N-terminus of CsgF. Taken together these data suggest that the structured region of CsgF, between residues 66 and 128, is involved in the proteins interaction with hIAPP and that upon interaction structural changes make the N-terminus less solvent exposed.

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in the protein misfolding, and metal imbalance may be part of TSE pathologies. PrPC is a combined Cu(II) and Zn(II) metal binding protein, where the main metal binding site is located in the octarepeat (OR) region. Here, we used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Upon metal binding, the OR region seems to adopt a transient antiparallel {beta}-sheet hairpin structure. Fluorescence spectroscopy data indicates that under neutral conditions, the OR region can bind both Cu(II) and Zn(II) ions, whereas under acidic conditions it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of both metal ions to the OR region results in formation of {beta}-hairpin structures. As formation of {beta}-sheet structures is a first step towards amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSEs.

The human prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-linked membrane-bound glycoprotein, containing two glycosylation sites. Human PrP is associated with a number of neurodegenerative diseases, called transmissible spongiform encephalopathies (TSE). Pathogenesis involves a structural conversion of the cellular form (PrPC), rich in -helical and random coil structure, into the scrapie form (PrPSc) characterized by parallel in register intermolecular {beta}-sheet conformation. To get a better understanding of this structural conversion, it is crucial to first characterize the non-pathogenic cellular isoform including all posttranslational modifications, like GPI-anchoring and native-like human glycosylation pattern. So far, studies on PrPC or PrPSc as well as the transition from one state to the other rely on non-native constructs of PrP studied far away from physiological conditions. We, therefore, established the expression of GPI-linked human PrP with close to native glycosylation pattern (native-like human PrP) using the eukaryotic LEXSY expression system in Leishmania tarentolae. This expression system has the added advantage that it allows for large-scale production of the native-like human PrP, which results in [~]1 mg purified protein per liter culture. Sedimentation velocity analysis and far-UV circular dichroism spectroscopy confirm the high structural homogeneity and monomeric native-like conformation of the purified GPI-anchored human PrP.

The SARS-CoV-2 main protease (Mpro) is a crucial enzyme responsible for the maturation of novel coronavirus, thus it serves as an excellent target for drug discovery. SARS-CoV-2 is found to have similarity with SARS-CoV, which showed conformational changes upon varying pH. There is no study till date on how pH change affect the conformtional flexibilty of SARS-CoV-2 Mpro, therefore, we attempt to find the effect of pH variation through constant pH molecular dynamics simulation studies. Protein is found to be most stable at neutral pH and as pH turns basic protein structure becomes most destabilized. Acidic pH also tends to change the structural properties of Mpro. Our study provides evidence that the flexibility of Mpro is pH dependent like SARS-CoV Mpro.

The search for peptides that can specifically bind to regulatory regions in DNA is a necessary step for creating drugs that can regulate gene expression. The study is dedicated to the peculiarities of binding of a model peptide, which carries an ionic self-complementary motif and can form amyloid-like fibrils [1], with model double-stranded DNAs. The stoichiometric ratios of the components of the complex were found using the retardation method in agarose gel. Using microscale thermophoresis, it was shown that the peptide in the amyloid-like state is capable of binding to model 45-bp double-stranded DNA, with a micromolar equilibrium dissociation constant. Using cryo-electron, transmission electron, and atomic force microscopy, the morphology of peptide-DNA complexes was studied. Using dynamic light scattering and nanoparticle tracking analysis, as well as small-angle neutron scattering, the spatial parameters of the resulting DNA-peptide complexes were characterized. Molecular dynamics simulations showed that the arginine side chains of the peptide are prone to interact with guanine nitrogenous bases. It was shown that the formation of peptide-dsDNA complexes interferes with the operation of restriction endonucleases that have guanine-cytosine pairs in the recognition center, which is consistent with the results of prediction of interaction sites obtained using computer modeling. The results of the work can be used in the development of peptides capable of interacting with functional regions of DNA, as well as in the development of new carriers for transfection of DNA constructs.

Structural characterization of the prion prone TAR DNA Binding protein (TDP)-43 has been challenging since its intrinsically disordered regions represents 15-30% of the total protein. TDP-43 is a nucleic acid binding protein with an N-terminal domain, two RNA Recognition Motifs (RRM1 and RRM2) and the C-terminal domain. In this study, we seek to define possible new targetable sites on the apo structure of TDP-43 RRM domains. To do so, we used molecular dynamic (MD) simulations on the NMR solved TDP-43RRM1-2 structure bound to RNA to predict the apo structure. Contact analysis of TDP-43 showed that while the integrity of the individual domains was maintained upon RNA removal, a decrease in interdomain contacts was observed. Moreover, we compared apo TDP-43 structures obtained from MD to AlphaFold 2 (AF2) predicted TDP-43 structures and found differences in loop regions. A Sitemap analysis identified five druggable sites for the RNA bound structure solved by NMR, while fewer sites were identified following MD simulations and AF2 predicted apo structures.

We here report a study of aggregation of Semaglutide at different temperatures, using resonance light scattering (RLS), fluorescence polarization and back-scattering techniques. Fluorescence emission spectra were obtained by exciting the samples at 275 nm and 295 nm, revealing a peak emission at 600 nm associated with the aggregation process. The size of the aggregates is around 100 nm according to back-scattering measurements. Two distinct thermal transitions were observed by RLS: the first melting point (Tm1) at 30{degrees}C and the second (Tm2) at 91{degrees}C, indicating changes in aggregation state. The fluorescence polarization revealed a fast rotational dynamics of the aggregates at Tm2, leading to greater depolarization of the emitted light. The structural organization of the Ozempic aggregates was studied using two dyes, Laurdan for lipid components and 1,8-ANS for protein component (GLP1). Thus, revealing a stable PEG-lipid core which hold the GLP1, increasing their exposition to the solvent. An enhanced FRET event inside the aggregates in presence of Fe2+ and Fe3+ was observed. We conclude that the PEG-lipid core plays a significant role in the aggregates structure stability, being a key to improve the biological activity of Ozempic. This methodology can be used to study similar aggregation constructs in the pharmaceutical industry.

Aggregation of intrinsically disordered proteins (IDPs) is the cause of various neu-rodegenerative diseases. Changes in solution pH can trigger IDP aggregation due to a shift in the IDP monomer population with a high aggregation propensity. Al-though there is experimental evidence that acidic pH promotes the compaction of IDP monomers, which subsequently leads to aggregation, the general mechanism is not clear. Using the IDP prothymosin- (proT), which is involved in multiple essential functions as a model system, we studied the pH effect on the conformational ensemble of proT and probed its role in aggregation using a coarse-grained IDP model and molecular dynamics simulations. We show that compaction in the proT dimension at low pH is due to the proteins collapse in the intermediate region (E41 - D80) rich in glutamic acid residues. Further, the {beta}-sheet content increases in this region upon pH change from neutral to acidic. We hypothesized that the conformations with high {beta}-sheet content could act as aggregation-prone (N*) states and nucleate the aggregation process. We validated our hypothesis by performing dimer simulations starting from N* and non-N* states. We show that simulations initiated using N* states as initial conformations form dimers within 1.5 s, whereas the non-N* states do not form dimers within this timescale. This study contributes to understanding the general principles of pH-induced IDP aggregation. The main result upon pH change from neutral to acidic, the intermediate region of proT is responsible for aggregation due to an increase in its {beta}-sheet forming propensity and forms the fibril core can be verified by experiments. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/497626v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@19971beorg.highwire.dtl.DTLVardef@fa98bforg.highwire.dtl.DTLVardef@422e5borg.highwire.dtl.DTLVardef@f18187_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transferrin is an attractive candidate for drug delivery due to its ability to cross the blood brain barrier. However, in order to be able to use it for therapeutic purposes, it is important to investigate how its stability depends on different formulation conditions. Combining high-throughput thermal and chemical denaturation studies with small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, it was possible to connect the stability of transferrin with its conformational changes. The release of iron induces opening of transferrin, which results in a negative effect on its stability. Presence of NaCl, arginine, and histidine leads to opening of the transferrin N-lobe and has a negative impact on the overall protein stability.\n\nStatement of significanceProtein-based therapeutics have become an essential part of medical treatment. They are highly specific, have high affinity and fewer off-target effects. However, stabilization of proteins is critical, time-consuming, and expensive, and it is not yet possible to predict the behavior of proteins under different conditions. The current work is focused on a molecular understanding of the stability of human serum transferrin; a protein which is abundant in blood serum, may pass the blood brain barrier and therefore with high potential in drug delivery. Combination of high throughput unfolding techniques and structural studies, using small angle X-ray scattering and molecular dynamic simulation, allows us to understand the behavior of transferrin on a molecular level.

Secreted apolipoprotein L1 (APOL1) is well known as an innate immune factor, protecting against African trypanosomiasis. The intracellular form has multiple functions, including regulating autophagy, intracellular vesicle trafficking, and ion channel activity. The APOL1 protein (G0) has two common variants (denoted G1 and G2) in the C-terminal region and are associated with a high risk of chronic kidney disease (CKD) and progression to end-stage kidney disease. Our previous studies using molecular modeling suggested that APOL1 G1 and G2 stabilize an autoinhibited state of the C-terminus, leading to impaired intracellular interactions with SNARE proteins. To characterize the structural consequence of kidney disease-associated APOL1 variants further, we assigned the C-terminal region proteins using 1H, 13C, 15N multidimensional nuclear magnetic resonance (NMR) spectra in solution in the presence of membrane mimetic dodecylphosphocholine micelles. We then derived models for the three-dimensional structure of APOL1-G0, and -G1 and -G2 variant C-terminal regions using the chemical shifts of the main chain nuclei followed by NMR relaxation measurements. The data suggest that changes in the three-dimensional structure of APOL1 C-terminal region induced by kidney disease-associated variants, not least the alteration of key sidechains and their interactions, could disrupt membrane association and the yet to be characterized protein-protein interactions including its binding partners, such as SNARE proteins. Such interactions could underlie the intracellular mechanisms that mediate the pathogenesis of CKD. In the future, one may try to reverse such structural and dynamics changes in the protein by designing agents that may bind and then mitigate APOL1 variant-associated CKD.

The most common age-related neurodegenerative disorder, Alzheimers disease, is clinically characterized by continuous neuronal loss resulting in loss of memory and dementia with no cure to date. Amyloid-{beta} (A{beta}) aggregates and tau protein are believed to be the causative agents of this pathogenesis. In the present study, we have investigated the effect of the Pro-Drug peptide (PDp) and its metabolites (-aspartyl & {beta}-aspartyl) on the A{beta} aggregates using atomistic molecular dynamics simulations. One of the key findings in our work is in the presence of -aspartyl as a ligand, the salt bridges which hold the N-terminals together are completely disrupted, thus setting the N-terminals free and exposed entirely to the solvent which can make the aggregation of A{beta} less severe. The efficiency of the ligands, which are responsible for the disruption of A{beta}, depends on the alignment and strength of the repulsive interactions. Besides repulsive interactions, we found that there is a need for hydrogen bonding, which acts as a support for the ligand to stay in the vicinity of the aggregate. Moreover, we have noticed that one of the metabolites, namely {beta}-aspartyl, formed more hydrogen bonds with the aggregate than the other ligands and had a different mode of action with the chains of A{beta} due to its unique flexible kink in the backbone.

Withdrawn reasonThe author has withdrawn the manuscript because additional results with complementary techniques are expected to complement the already gathered data and thus, having a more complete overview of the impact of several conditions of interest on the S100A9 fibrillation processes. Therefore, the author do not wish this work to be cited as reference for the project. If you have any questions, please contact with the author.

Biophysical techniques such as Isothermal Calorimetry (ITC) and Surface Plasmon Resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants. Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2-CMP + RNASE and BH3I-1 + hBCLXL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately.

In the present study, we measured the changes in the higher-order structure of genomic DNA molecules in the presence of alcohols through single-DNA observation by use of fluorescence microscopy, with particular focus on the different effects of 1-propanol and 2-propanol. The results showed that, with an increasing concentration of 1-propanol, DNA exhibits reentrant conformational transitions from an elongated coil to a folded globule, and then to an unfolded state. On the other hand, with 2-propanol, DNA exhibits monotonous shrinkage into a compact state. Thus, DNA molecules are more effectively condensed/precipitated with 2-propanol than with 1-propanol. The propanol isomers also had different effects on the changes in the secondary structure of DNA, as revealed by circular dichroism (CD) measurements. With 1-propanol, DNA maintains a B-form secondary structure. An A-like conformation appears with the addition of 2-propanol.\n\nSTATEMENT OF SIGNIFICANCECurrently, 2-propanol has most often been used as the solvent to extract and purify genomic DNA molecules from living cells, according the protocols in molecular biology and biochemistry. Unfortunately, the reason why usage of 2-propanol is recommended instead of ethanol and 1-propanol has never been explained in a clear manner. We believe that the new insight based on chemical physics point of view would play an important role for the development of current chemical procedures/treatments adapted on an empirical basis.

The intrinsically disordered microtubule-associated protein tau is known for its aberrant aggregation into neurofibrillary tangles as found in neuropathologies such as Alzheimers disease. This study compares three N-terminal isoforms of mutant R5L and of wild type tau to investigate how this mutation and the length of the projection domain affects aggregation behavior. Tau polymers in vitro were examined using atomic force microscopy imaging to compare tau filament lengths and morphologies. In a complementary analysis, the total amount of polymerization was analyzed using a Thioflavin S assay. We observed that the R5L mutation has a greater impact on filament length in shorter N-terminal isoforms of tau, whereas in longer N-terminal isoforms the mutation impacts the total amount of tau aggregation. These observations suggest that the R5L mutation affects the kinetic nucleation-elongation pathway of tau fibrillization, where the mutant impacts polymer nucleation in 2N and 1N isoforms, but has a more significant impact on elongation in the 0N isoform.

Synucleinopathies, including Parkinsons disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA) have the same hallmark pathologic feature of misfolded -synuclein protein accumulation in the brain. PD patients who carry -syn hereditary mutations tend to have an earlier onset and more severe clinical symptoms and pathology than sporadic PD patients who carry wild-type (WT) -syn. Therefore, revealing the structural effect of -syn hereditary mutations on the wild-type fibril structure can help us understand synucleinopathies structural basis. Here, we present a 3.38 [A] cryo-electron microscopy structure of -synuclein fibrils containing the hereditary A53E mutation. The A53E fibril is symmetrically composed of two protofilaments, as are many other synucleopathic structures - including WT. Interestingly, the interface between the protofilaments in A53E has significantly less buried surface area than all other documented fibril structures of -syn and its other mutants. The A53E fibril also exhibits slower formation/growth in in vitro fibrillation experiment compared to other mutants. This implies that the structural differences - both in the protofilament and between each protofilament of A53E - change the aggregation mechanism, or in the least, its kinetics of formation. These differences influence the molecular characteristics of each fibril mutant and likely plays a macro-scale role in progressing one clinical pathology over another.

The structure-function relationship of the riboswitch is governed mainly by two factors, ligand binding and temperature. Most of the experimental studies shed light on structural dynamics and gene regulation function of Adenine riboswitch from the aspect of ligand instead of temperature. Two unliganded Adenine riboswitch conformations (apoA and apoB) from the thermophile Vibrio vulnificus draw particular attention to the Biophysics research community due to their diverse and polymorphic structures. Ligand-free apoB Adenine riboswitch conformation is not able to interact with the ligand whereas ligand-free apoA Adenine riboswitch conformation adopts ligand-receptive form. The interconversion between apoA and apoB conformation is temperature-dependent and thermodynamically controlled. Therefore Adenine riboswitch is called a temperature sensing RNA. The molecular mechanism underlying the thermosensitivity of ligand free Adenine riboswitch is not well known. Hence it is essential to explore temperature-induced conformational dynamics of unliganded Adenine riboswitch. In this research work I make an attempt to examine conformational stability and order of apoA with respect to apoB Adenine riboswitch aptamer using conformational thermodynamics derived from all-atom molecular dynamics trajectories in the temperature range 283K-400K. The changes in conformational free energy and entropy of conformational degrees of freedom like pseudo-torsion angle and {theta} are computed. RMSD, RMSF, RG, principal component analysis, hydrogen bonding interaction, and conformational thermodynamics data demonstrate that conformational stability and order of apoA with respect to apoB adenine riboswitch conformation is significant at 293K and 303K. The temperatures corresponding to the conformational order and stability of apoA adenine riboswitch with respect to apoB adenine riboswitch whole aptamer are shown in descending order 293K[~]303K> 313K[~]283K>373K>323K. The topological and conformational changes related to hydrogen bonding reorganization occur mostly at temperature 323K and 400K. Ligand unbound adenine riboswitch is sensitive to heat and may be inactivated at temperatures 323K and 400K. HighlightsO_LIMolecular Dynamics Simulation of ligand unbound two different conformations of adenine riboswitch with the same sequence at different temperatures are performed. C_LIO_LIAt low temperatures (293K and 303K) the conformational stability of apoA is more pronounced compared to apoB adenine riboswitch. C_LIO_LIThermal stability of apoA compared to apoB adenine riboswitch is the least at 323K temperature. C_LIO_LIAt higher temperature (373K) and lower temperature (283K) apoA adenine riboswitch exerts moderate conformational stability. C_LIO_LIThe nucleotides from stem2 and junction (J1/2, J2/3) regions of adenine riboswitch contribute the most to the conformational stability and order. C_LI