Native Conformational State Research Articles

Structural Dynamics of SNARE Complex Assembly in the Ribbon Synapses Observed by smFRET.

Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique for studying the structural dynamics of protein molecules or detecting interactions between protein molecules in real time. Due to the high sensitivity in spatial and temporal resolution, smFRET can decipher sub-populations within heterogeneous native state conformations, which are generally lost in traditional measurements due to ensemble averaging. In addition, the single-molecule reconstitution allows protein molecules to be observed for an extensive period of time and can recapitulate the geometry of the cellular environment to retain biological function. Here we provide a detailed method of using smFRET to monitor the conformational dynamics of syntaxin-3b from the ribbon synapses during assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.

Exploiting Copaifera salikounda compounds as treatment against diabetes: An insight into their potential targets from a computational perspective

Accumulating evidence has shown that medicinal plants have been exploited for treatment purposes since time immemorial. Thus, this study investigated the mitigating potentials of the ligands; n-hexadecanoic acid, 9-octadecenoic acid and octadecanoic acid from Copaifera salikounda seed pond extract reported to have antidiabetic potentials in our previous study using computational techniques. Fatty acid-binding protein 4 (FABP4) and peroxisome proliferator-activated receptor alpha (PPARα) were identified as potential receptors. Both molecular docking and Estimated ΔGbind revealed that each ligand exhibited high binding affinity to the respective proteins; this is quite sufficient to be termed favourable. A critical examination of the type and the nature of binding interactions and energy contributions have identified Arg106, Arg126 and Tyr128 in FABP4 and Gln277, Ser280, Tyr314, His440 and Tyr464 in PPARα as consistently being responsible for the binding interactions and stabilizations of each ligand to the individual proteins. The establishment of hydrogen bonding type of interaction and activity between the carboxylic acid moieties of these ligands and these crucial/unique residues goes further to buttress our assertion. A general study of the conformational state of these protein via RMSF and PCA plots goes further validate the observed structural trends wherein the presence of ligands induced seemly structural rigidity. In depth structural stability investigations went further to reveal that the 3D structures of these protein didn’t deviate from it known native conformational stable state when bound with these ligands. Our findings indicate that the ligands have considerable inhibitory action against FABP4 and PPARα corroborating the reported antidiabetic potential of the extract.

Evolutionary Couplings and Molecular Dynamic Simulations Highlight Details of GPCRs Heterodimers' Interfaces.

A growing body of evidence suggests that only a few amino acids ("hot-spots") at the interface contribute most of the binding energy in transient protein-protein interactions. However, experimental protocols to identify these hot-spots are highly labor-intensive and expensive. Computational methods, including evolutionary couplings, have been proposed to predict the hot-spots, but they generally fail to provide details of the interacting amino acids. Here we showed that unbiased evolutionary methods followed by biased molecular dynamic simulations could achieve this goal and reveal critical elements of protein complexes. We applied the methodology to selected G-protein coupled receptors (GPCRs), known for their therapeutic properties. We used the structure-prior-assisted direct coupling analysis (SP-DCA) to predict the binding interfaces of A2aR/D2R, CB1R/D2R, A2aR/CB1R, 5HT2AR/D2R, and 5-HT2AR/mGluR2 receptor heterodimers, which all agreed with published data. In order to highlight details of the interactions, we performed molecular dynamic (MD) simulations using the newly developed AWSEM energy model. We found that these receptors interact primarily through critical residues at the C and N terminal domains and the third intracellular loop (ICL3). The MD simulations showed that these residues are energetically necessary for dimerization and revealed their native conformational state. We subsequently applied the methodology to the 5-HT2AR/5-HTR4R heterodimer, given its implication in drug addiction and neurodegenerative pathologies such as Alzheimer's disease (AD). Further, the SP-DCA analysis showed that 5-HT2AR and 5-HTR4R heterodimerize through the C-terminal domain of 5-HT2AR and ICL3 of 5-HT4R. However, elucidating the details of GPCR interactions would accelerate the discovery of druggable sites and improve our knowledge of the etiology of common diseases, including AD.

Probing the Energy Landscape of Spectrin R15 and R16 and the Effects of Non-native Interactions.

Understanding the details of a protein folding mechanism can be a challenging and complex task. One system with an interesting folding behavior is the α-spectrin domain, where the R15 folds three-orders of magnitude faster than its homologues R16 and R17, despite having similar structures. The molecular origins that explain these folding rate differences remain unclear, but our previous work revealed that a combined effect produced by non-native interactions could be a reasonable cause for these differences. In this study, we explore further the folding process by identifying the molecular paths, metastable states, and the collective motions that lead these unfolded proteins to their native state conformation. Our results uncovered the differences between the folding pathways for the wild-type R15 and R16 and an R16 mutant. The metastable ensembles that speed down the folding were identified using an energy landscape visualization method (ELViM). These ensembles correspond to similar experimentally reported configurations. Our observations indicate that the non-native interactions are also associated with secondary structure misdocking. This computational methodology can be used as a fast, straightforward protocol for shedding light on systems with unclear folding or conformational traps.

Novel Autoantibodies in Idiopathic Small Fiber Neuropathy.

ObjectiveSmall fiber neuropathy (SFN) is clinically and etiologically heterogeneous. Although autoimmunity has been postulated to be pathophysiologically important in SFN, few autoantibodies have been described. We aimed to identify autoantibodies associated with idiopathic SFN (iSFN) by a novel high‐throughput protein microarray platform that captures autoantibodies expressed in the native conformational state.MethodsSera from 58 SFN patients and 20 age‐ and gender‐matched healthy controls (HCs) were screened against >1,600 immune‐related antigens. Fluorescent unit readout and postassay imaging were performed, followed by composite data normalization and protein fold change (pFC) analysis. Analysis of an independent validation cohort of 33 SFN patients against the same 20 HCs was conducted to identify reproducible proteins in both cohorts.ResultsNine autoantibodies were screened with statistical significance and pFC criteria in both cohorts, with at least 50% change in serum levels. Three proteins showed consistently high fold changes in main and validation cohorts: MX1 (FC = 2.99 and 3.07, respectively, p = 0.003, q = 0.076), DBNL (FC = 2.11 and 2.16, respectively, p = 0.009, q < 0.003), and KRT8 (FC = 1.65 and 1.70, respectively, p = 0.043, q < 0.003). Further subgroup analysis into iSFN and SFN by secondary causes (secondary SFN) in the main cohort showed that MX1 is higher in iSFN compared to secondary SFN (FC = 1.61 vs 0.106, p = 0.009).InterpretationNovel autoantibodies MX1, DBNL, and KRT8 are found in iSFN. MX1 may allow diagnostic subtyping of iSFN patients. ANN NEUROL 2022;91:66–77

Hybrid hydrogel reactor with metal–organic framework for biomimetic cascade catalysis

It has always been a goal for immobilized enzymes to work under their native conformational states while possessing robust stabilities in harsh environments. Hydrogel is a promising enzyme matrix because of its cell-like microenvironment and porous internal structure to preserve the conformations of enzymes, maximizing catalytic efficiency. However, the challenges in low chemical tolerance and poor mechanical strength remained. Herein, a unique organic/inorganic hybrid hydrogel system was fabricated through the interfacial mineralization of metal–organic frameworks (MOFs) on hydrogel microsphere for processing biomimetic cascade reaction. The combination of hydrogel and MOFs layer is synergistic. The porous hydrogel network offers a biocompatible space for encapsulated enzymes to eliminate their undesirable restriction and chemical interactions with hosts, allowing the enzymes to work under native states for benefiting high activity, while the inorganic MOFs layer protects the enzymes from harsh environments to endow the hybrid reactor with robust stabilities. Attractively, a high enzyme tolerance against polar organic solvents was approached in the hybrid reactor. In addition, by the spatial arrangement of different enzymes in inner hydrogel and outer MOFs layer, the hybrid reactor can also function as a multi-compartmental structure for performing incompatible enzyme reactions to give distinguished reaction fluxes and product outputs, as occurred in natural cells.

Exploring the Binding Interaction of Raf Kinase Inhibitory Protein With the N-Terminal of C-Raf Through Molecular Docking and Molecular Dynamics Simulation.

Protein-protein interactions are indispensable physiological processes regulating several biological functions. Despite the availability of structural information on protein-protein complexes, deciphering their complex topology remains an outstanding challenge. Raf kinase inhibitory protein (RKIP) has gained substantial attention as a favorable molecular target for numerous pathologies including cancer and Alzheimer’s disease. RKIP interferes with the RAF/MEK/ERK signaling cascade by endogenously binding with C-Raf (Raf-1 kinase) and preventing its activation. In the current investigation, the binding of RKIP with C-Raf was explored by knowledge-based protein-protein docking web-servers including HADDOCK and ZDOCK and a consensus binding mode of C-Raf/RKIP structural complex was obtained. Molecular dynamics (MD) simulations were further performed in an explicit solvent to sample the conformations for when RKIP binds to C-Raf. Some of the conserved interface residues were mutated to alanine, phenylalanine and leucine and the impact of mutations was estimated by additional MD simulations and MM/PBSA analysis for the wild-type (WT) and constructed mutant complexes. Substantial decrease in binding free energy was observed for the mutant complexes as compared to the binding free energy of WT C-Raf/RKIP structural complex. Furthermore, a considerable increase in average backbone root mean square deviation and fluctuation was perceived for the mutant complexes. Moreover, per-residue energy contribution analysis of the equilibrated simulation trajectory by HawkDock and ANCHOR web-servers was conducted to characterize the key residues for the complex formation. One residue each from C-Raf (Arg398) and RKIP (Lys80) were identified as the druggable “hot spots” constituting the core of the binding interface and corroborated by additional long-time scale (300 ns) MD simulation of Arg398Ala mutant complex. A notable conformational change in Arg398Ala mutant occurred near the mutation site as compared to the equilibrated C-Raf/RKIP native state conformation and an essential hydrogen bonding interaction was lost. The thirteen binding sites assimilated from the overall analysis were mapped onto the complex as surface and divided into active and allosteric binding sites, depending on their location at the interface. The acquired information on the predicted 3D structural complex and the detected sites aid as promising targets in designing novel inhibitors to block the C-Raf/RKIP interaction.

Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM-based single-cell force spectroscopy

Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin–cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins’ native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins’ homophilic bonds.

Detection of putative autoantibodies in systemic lupus erythematous using a novel native-conformation protein microarray platform.

Conventional immunoassays detect autoantibodies related to systemic lupus erythematosus (SLE) via recognition of epitopes on autoantigens expressed in their denatured rather than native conformational state, casting difficulty in evaluating the genuine pathogenicity of the autoantibodies. We aimed to use a novel high-throughput protein microarray platform to identify autoantibodies against native autoantigens in SLE sera. Sera from SLE patients and those of gender-, age-, and ethnicity-matched healthy controls (HC) were screened against more than 1,600 immune-related antigens of native conformation. The relative fluorescent unit readout from post-assay imaging were subjected to bioinformatics pre-processing and composite normalization. A penetrance fold change (pFC) analysis between SLE and HC samples shortlisted 50 autoantigens that were subjected to an unsupervised cluster analysis. Correlations between the pFC of putative autoantigens and clinical parameters including SLE disease activity index (SLEDAI-2K) and recent SLE flares were explored. 381 autoantigens were identified when 15 SLE and 15 HC serum samples were compared. The top 20 autoantigens which elicited autoantibody responses in SLE sera filtered based on the highest pFC were further analyzed. Autoantigens which the putative autoantibodies reacted against are those involved in chromatin organization such as DEK, regulation of transcription activity including REOX4 and ELF4, and negative regulation of NFkB activity such as TRIB3. Additionally, the pFC of these autoantibodies significantly and positively correlated with SLEDAI-2K and recent SLE flares. A high-throughput protein microarray platform allows detection and quantification of putative lupus-related autoantibodies which are of potential pathophysiological and prognostic significance in SLE patients.

Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins.

Membranes proteins make up more than 60% of current drug targets and account for approximately 30% or more of the cellular proteome. Access to this important class of proteins has been difficult due to their inherent insolubility and tendency to aggregate in aqueous solutions. Understanding membrane protein structure and function demands novel means of membrane protein production that preserve both their native conformational state as well as function. Over the last decade, cell-free expression systems have emerged as an important complement to cell-based expression of membrane proteins due to their simple and customizable experimental parameters. One approach to overcome the solubility and stability limitations of purified membrane proteins is to support them in stable, native-like states within nanolipoprotein particles (NLPs), aka nanodiscs. This has become common practice to facilitate biochemical and biophysical characterization of proteins of interest. NLP technology can be easily coupled with cell-free systems to achieve functional membrane protein production for this purpose. Our approach involves utilizing cell-free expression systems in the presence of NLPs or using co-translation techniques to perform one-pot expression and self-assembly of membrane protein/NLP complexes. We describe how cell-free reactions can be modified to render control over nanoparticle size and monodispersity in support of membrane protein production. These modifications have been exploited to facilitate co-expression of full-length functional membrane proteins such as G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In particular, we summarize the state of the art in NLP-assisted cell-free coexpression of these important classes of membrane proteins as well as evaluate the advances in and prospects for this technology that will drive drug discovery against these targets. We conclude with a prospective on the use of NLPs to produce as well as deliver functional mammalian membrane-bound proteins for a range of applications.

The Effects of Acetylation on Beta‐2‐Microglobulin on Amyloid Formation

Beta‐2‐Microglobulin (B2m) is a small 99 residue globular protein that is a part of the type I major histocompatibility complex found on all nucleated cells. It is shed from the surface of cells into the bloodstream as cells undergo apoptosis then is degraded and excreted from the body in people with normal functioning kidneys. However, B2m is not effectively removed from the blood by hemodialysis causing systemic problems for patients in end‐stage kidney disease (ESKD). B2m serum levels in patients with ESKD are up to 60 times greater than healthy individuals. Dialysis related amyloidosis (DRA), is a disease that is often experienced by those with ESKD. DRA results from the accumulation of misfolded B2m and is characterized by carpal tunnel like symptoms coupled with chronic joint inflammation. DRA is a result of the deposition of misfolded B2m in the musculoskeletal system due to B2m amyloid plaque formation. However, the increased concentration of B2m alone is not sufficient to trigger the pathogenic amyloidosis at physiological pH. Thus, a secondary factor must be acting as the trigger of misfolding. This study is investigating the effect of acetylation as a non‐natural post translational modification on B2m. Acetylation is a practicable suspect as aspirin consumption is known to acetylate a variety of proteins non‐specifically. The acetylation of lysines would alter the overall charge and possibly disrupt ability of B2m to maintain its native state conformation thus increasing the aggregation propensity of B2m. We are primarily testing the susceptibility of B2m to acetylation via protocol established for acetylation in bovine carboxy anhydrase. Additionally, growth of the amyloid fibrils of the acetylated B2m will be monitored with thioflavin‐T fluorescence to assess the effects of lysine acetylation on B2m amyloid formation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of folding-sensitive nanobodies as tools to study the expression and quality of protein particle immunogens

Vaccination is as one of the most beneficial biopharmaceutical interventions against pathogens due to its ability to induce adaptive immunity through targeted activation of the immune system. Each vaccine needs a tailor-made set of tests in order to monitor its quality throughout the development and manufacturing. The analysis of the conformational state of protein nanoparticles is one of the key steps in vaccine quality control. The enzyme lumazine synthase from Brucella spp. (BLS) acts as a potent oral and systemic immunogen. BLS has been used as a carrier of foreign peptides, protein domains and whole proteins, serving as a versatile platform for vaccine engineering purposes. Here, we show the generation and characterization of four families of nanobodies (Nbs) which only recognize BLS in its native conformational state and that bind to its active site. The present results support the use of conformation-sensitive Nbs as molecular probes during the development and production of vaccines based on the BLS platform. Finally, we propose Nbs as useful molecular tools targeting other protein scaffolds with potential applications in nano-and biotechnology.

Encapsulation driven conformational changes in n-alkanes inside a hydrogen-bonded supramolecular cavitand assembly

Molecular chaperones are known to assist proteins in achieving their native state conformations by providing them a confined environment through encapsulation and altering their free energy landscapes. Instances of structural rearrangements in molecules confined to narrow nano-dimensional spaces are often encountered in vivo where the intermolecular interactions play a decisive role in determining the molecular conformations. As a simple analogue, we model a system of n-alkanes encapsulated into a supramolecular host cavitand forming a capsular host-guest assembly of volume less than half a cubic nanometer. Here, we provide a detailed insight of process of host-induced conformational changes in guests by exploring the conformational dynamics. Interestingly, the conformational analysis reveals that the host apart from being able to fold some of the guests into coiled conformations also unfolds a few of the guests while rearranging its hydrogen-bonded network.

Metal Induced Conformational Changes of Alpha‐Synuclein and the Role of Ambient Oxygen

Hypotheses surrounding the native conformational states of α‐synuclein (αS), which is known to be a key player in Parkinson's Disease (PD) pathology, have evolved in recent years to adopt both a monomeric and tetrameric form. Traditionally, the conformation of αS was described as an intrinsically disordered monomer that aggregates to form β‐sheet fibrils, potentially through oligomeric intermediates. Upon interaction with membranes, this monomeric conformation of αS is known to shift from its dynamic unfolded state to α‐helical. Tetrameric αS has been characterized as both α‐helical and aggregation resistant, likely a passive storage form of the protein. The pathological conformation of αS remains controversial but each functional fold and dysfunctional misfold must originate from the monomeric protein. Membrane‐protein interactions are thought to facilitate the transition of monomeric αS to the α‐helical tetramer due to the similarity in folding patterns.Physiological levels of cerebral biometals, such as copper and iron, change in various regions of the brain with aging and with advanced disease states. Metal dyshomeostasis has long been linked to PD but most of the work predated the discovery of the tetramer and the acceptance of αS as being post‐translationally acetylated at the N‐terminus. The latter has shown biophysical variation in regards to both αS conformational dynamics and metal coordination. The presented research will highlight metal‐induced changes in the folding pattern of αS and the distinguishable differences observed between various metal oxidation states. Unique changes are observed in the protein secondary structure and the hydrodynamic radius upon the introduction of stoichiometric quantities of individual metal ions based on circular dichroism and dynamic light scattering analyses respectively. Under oxidatively stressed conditions, metal‐bound αS can promote the generation of reactive oxygen species. Immunoblotting techniques have revealed that metal‐bound αS can follow distinct pathways towards toxic oligomers or β‐sheet fibrils depending on the metal. Furthermore, a clear involvement of ambient oxygen in the production of post‐translational modifications has been observed. Such metal‐induced conformational changes result in altered membrane interactions, which could have physiological implications. Taken together, these results contribute to the ongoing conversation within the field about the role of metals and αS in PD progression.Support or Funding InformationSupported by Virginia Commonwealth University, College of Humanities &amp; Sciences

Finding Protein Folding Funnels in Random Networks

Many attempts have been made for understanding protein folding dynamics using network representations of folding pathway. For example, the Markov state model has been widely used in analyzing the transitions between protein conformations of trajectories obtained by molecular dynamics simulations.In the present work, I also focus on the network structures of protein folding dynamics, but from a somewhat different perspective. Currently most widely accepted theoretical framework of the protein folding is so-called funnel picture, which states that the number of conformations decreases as the energy lowers from the denatured state towards the native state. In other words, the energy landscapes of proteins have been designed through Darwinian evolution so that proteins readily fold to their native states. A question I would like to ask here is “How rare are such folding-friendly funnel structures?”.As a first attempt to answer this question, I introduce a random network model with each nodes assigned a random energy. Nodes represent the metastable conformation ensembles. One of the nodes is considered as denatured state and another the folded state. Other nodes represent the intermediate conformation ensembles between them. Edge connecting the nodes are possible transitions between the nodes. The network structure is assumed to be determined by the native state conformation, and the energy assignment is considered to reflects the amino-acid sequence. We then define the ideal funnel structure for this model as follows: Starting from the denatured state, if all the paths connecting the nodes in energy-lowering direction reach the folded state, then such a network is in a ideal funnel structure. The task now is to compute the probability that the ideal funnel is realized for a given network by changing the assignment of energy.We generated 1000 random networks for number of nodes from 8 to 16. The probability is widely distributed from network to network, and close to the log-normal distribution . This fact means that there are a small number of networks that are robust against the mutation. The funnel structure becomes rarer exponentially as the protein becomes longer.

AFM-Based Single Molecule Techniques: Unraveling the Amyloid Pathogenic Species.

BackgroundA wide class of human diseases and neurodegenerative disorders, such as Alzheimer’s disease, is due to the failure of a specific peptide or protein to keep its native functional conformational state and to undergo a conformational change into a misfolded state, triggering the formation of fibrillar cross-β sheet amyloid aggregates. During the fibrillization, several coexisting species are formed, giving rise to a highly heterogeneous mixture. Despite its fundamental role in biological function and malfunction, the mechanism of protein self-assembly and the fundamental origins of the connection between aggregation, cellular toxicity and the biochemistry of neurodegeneration remains challenging to elucidate in molecular detail. In particular, the nature of the specific state of proteins that is most prone to cause cytotoxicity is not established.Methods:In the present review, we present the latest advances obtained by Atomic Force Microscopy (AFM) based techniques to unravel the biophysical properties of amyloid aggregates at the nanoscale. Unraveling amyloid single species biophysical properties still represents a formidable experimental challenge, mainly because of their nanoscale dimensions and heterogeneous nature. Bulk techniques, such as circular dichroism or infrared spectroscopy, are not able to characterize the heterogeneity and inner properties of amyloid aggregates at the single species level, preventing a profound investigation of the correlation between the biophysical properties and toxicity of the individual species.Conclusion:The information delivered by AFM based techniques could be central to study the aggregation pathway of proteins and to design molecules that could interfere with amyloid aggregation delaying the onset of misfolding diseases.

Sub-Millisecond Unfolding Kinetic Spectra Reveals Intermediate Transitions

The formation of stable partially folded structures on the folding pathway of a protein plays an important role for its correct function. Intermediate states mediate protein (sub-) domain folds to reach the most stable native conformational state. Serious diseases can be triggered if proteins are incorrectly folded stabilizing misfolded conformational states. Currently, no single biophysical tool exist that could provide the overall map all conformational transitions during fondling or unfolding kinetics. Here, we used the model enzyme lysozyme from the phage T4 (T4L), a simple two-subdomain protein, from which is known from prior NMR and stopped-flow experiments that it unfolds via at least two intermediate states. To complete the kinetic gap, we created a library of double mutants of the cysteine-free pseudo-wild type by inserting an unnatural amino acid and a cysteine mutation. We proceeded to site-specifically labeled them using orthogonal chemistry with a Forster resonance energy transfer (FRET) dye pair to build up a network of distance restrains spanning the enzyme. The unfolding process was monitored using traditional ensemble methods like CD- spectroscopy and fluorescence spectroscopic methods (ensemble time correlated single photon counting, single molecule fluorescence spectroscopy, (filtered) fluorescence correlation spectroscopy). Single molecule high precision FRET, with its high temporal and spatial resolution, covering over seven orders of magnitude on time, enables us to determine equilibrium transition rates between conformational states in denatured conditions. By using a priori knowledge from earlier studies, we are able to assign the formerly unknown rates to the folding and unfolding intermediate formation steps. The previously reported millisecond process, belonging to the interconversion of both intermediates, can be observed in our single-molecule multiparameter fluorescence detection histograms.

Preparation of hydrocarbon/fluorocarbon double-chain phospholipid polymer brusheson polyurethane films by ATRP

To fabricate artificial biomembrane mimicking cell surfaces, hydrocarbon/fluorocarbon double-chain phospholipid macromonomer was grafted on polyurethane (PU) film surfaces by surface-initiated atom transfer radical polymerization (SI-ATRP). The surface structures of modified PU film surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and water contact angle measurement. The results indicate that initiator densities on these polymer film surfaces have a significant impact on graft polymerization of this fluorocarbon phospholipid macromonomer. The phospholipid polymer brushes grafted on PU film surfaces could self-assemble into biomimetic membranes under water environment, as demonstrated by liquid/liquid static contact angle measurement, atomic force spectroscopy (AFM), and attenuated total reflectance Fourier transform infrared (ATR-FTIR). These biomimetic membranes could maintain water within them as the “surrounding” water. Such would be favorable condition for the preservation of native conformational state of proteins and cell membranes. This work provides a new approach to fabricate biomimetic membranes on biomaterials surfaces.

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0M TMAO increased the free energy change (ΔG0) significantly from 2.1 to 3.8kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.

Characterization of the near native conformational states of the SAM domain of Ste11 protein by NMR spectroscopy.

The sterile alpha motif or SAM domain is one of the most frequently present protein interaction modules with diverse functional attributions. SAM domain of the Ste11 protein of budding yeast plays important roles in mitogen-activated protein kinase cascades. In the current study, urea-induced, at subdenaturing concentrations, structural, and dynamical changes in the Ste11 SAM domain have been investigated by nuclear magnetic resonance spectroscopy. Our study revealed that a number of residues from Helix 1 and Helix 5 of the Ste11 SAM domain display plausible alternate conformational states and largest chemical shift perturbations at low urea concentrations. Amide proton (H/D) exchange experiments indicated that Helix 1, loop, and Helix 5 become more susceptible to solvent exchange with increased concentrations of urea. Notably, Helix 1 and Helix 5 are directly involved in binding interactions of the Ste11 SAM domain. Our data further demonstrate that the existence of alternate conformational states around the regions involved in dimeric interactions in native or near native conditions.