Model Protein System Research Articles

Surfactants modify the torsion properties of proteins: a single molecule study

Surfactants are widely used in diagnostic assays to prevent protein aggregation and non-specific adsorption at surfaces. Here, a single molecule magnetic torque tweezers study is reported, aiming to quantify surfactant-induced changes in the torsional flexibility of a protein model system: protein-G-immunoglobulin G (IgG) attached to a glass surface. The influences of Sodium Dodecyl Sulphate (SDS) and Polysorbate 20 (Tween 20) on the protein pair have been investigated. The proteins were exposed to the surfactants at concentrations relative to the Critical Micelle Concentration (CMC), namely 0.1× CMC, 1× CMC and 10× CMC. Both surfactants increase the torsional flexibility of the protein-G-IgG complex. Tween 20 is most effective at increasing the torsional flexibility of the complex at the surface while SDS is more effective at dissociating the protein bonds. Tweezer data on the IgG-IgG protein pair show no influence of Tween 20 on the torsional flexibility. Furthermore, temperature dependent near-UV and far-UV Circular Dichroism (CD) data at 10× CMC show that Tween 20 does not significantly alter the secondary and tertiary structure of both protein-G and IgG while SDS does. These results provide evidence that both the mechanical properties of the protein structure and the interaction between proteins can alter the torsional rigidity measured with magnetic torque tweezers. This study shows for the first time the ability to use magnetic torque tweezers as a probe for surfactant-induced changes in proteins at a single molecule level.

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3β-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions.

Role of zwitterions in kindling fluorescent protein photochemistry.

Kindling fluorescent protein (KFP), one of the chronologically first members of photoswitchable colored proteins from the green fluorescent protein (GFP) family, increasingly attracts efforts from experimental and theoretical sides. Ambiguous conclusions in solving puzzles of photochemistry of KFP and of its parent natural protein asFP595 are partially explained by lack of reliable theoretical data on chromophore properties in the electronically excited state. We report the results of state-of-the-art quantum chemistry calculations of the structure and energy of the KFP chromophore, 2-acetyl-,4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone (AHBMI), both in the ground and excited states. Ground state equilibrium structures of anionic and zwitterionic protonation states of AHBMI were computed by the conventional MP2 method while excited state structures were characterized by the extended multireference perturbation theory method XMCQDPT2 including optimization of geometry parameters at this level. In particular, the computational results demonstrate that the basicity of the N2 nitrogen atom of the imidazolinone ring should noticeably increase upon electronic excitation, thus affecting excited state proton transfer events in proteins. The results of these simulations as well as of quantum mechanical-molecular mechanical (QM/MM) calculations for model protein systems evidence that KFP conformations with the zwitterionic chromophore are hardly expected to occur in the ground state, but may be populated upon excitation.

Physiological functions of MTA family of proteins.

Although the functional significance of the metastasic tumor antigen (MTA) family of chromatin remodeling proteins in the pathobiology of cancer is fairly well recognized, the physiological role of MTA proteins continues to be an understudied research area and is just beginning to be recognized. Similar to cancer cells, MTA1 also modulates the expression of target genes in normal cells either by acting as a corepressor or coactivator. In addition, physiological functions of MTA proteins are likely to be influenced by its differential expression, subcellular localization, and regulation by upstream modulators and extracellular signals. This review summarizes our current understanding of the physiological functions of the MTA proteins in model systems. In particular, we highlight recent advances of the role MTA proteins play in the brain, eye, circadian rhythm, mammary gland biology, spermatogenesis, liver, immunomodulation and inflammation, cellular radio-sensitivity, and hematopoiesis and differentiation. Based on the growth of knowledge regarding the exciting new facets of the MTA family of proteins in biology and medicine, we speculate that the next burst of findings in this field may reveal further molecular regulatory insights of non-redundant functions of MTA coregulators in the normal physiology as well as in pathological conditions outside cancer.

Protein Encapsulation via Polypeptide Complex Coacervation.

Proteins have gained increasing success as therapeutic agents; however, challenges exist in effective and efficient delivery. In this work, we present a simple and versatile method for encapsulating proteins via complex coacervation with oppositely charged polypeptides, poly(l-lysine) (PLys) and poly(d/l-glutamic acid) (PGlu). A model protein system, bovine serum albumin (BSA), was incorporated efficiently into coacervate droplets via electrostatic interaction up to a maximum loading of one BSA per PLys/PGlu pair and could be released under conditions of decreasing pH. Additionally, encapsulation within complex coacervates did not alter the secondary structure of the protein. Lastly the complex coacervate system was shown to be biocompatible and interact well with cells in vitro. A simple, modular system for encapsulation such as the one presented here may be useful in a range of drug delivery applications.

Crystallographic fragment screening: challenges, opportunities, lessons learned

Fragment-based approaches are now routinely applied for lead development in pharmaceutical drug research. Usually, a small but well selected library of low molecular weight compounds is pre-screened by biochemical or biophysical methods such as surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) or thermal shift assay; often followed for promising hit candidates by X-ray crystallography. We designed a small fragment library consisting of 364 compounds that is not strictly compliant to the otherwise often followed Astex rule of three for fragment library composition.[1] Thereafter, our library was validated on the pepsin-like aspartyl protease endothiapepsin, which serves as a model system for proteins that are involved in serious diseases such as malaria (plasmepsins), hypertension (renin) and Alzheimer's disease (ß-secretase) and therefore, is a valid target for further drug development. Due to the small size of fragments, they frequently exhibit only low affinity to the applied target protein and thus are often hard to detect in any screening approach, reflected in little overlap between different screening methods. After initial screening, we decided to validate the entire library by X-ray crystallography, which requires a steady supply of crystals, reproducible soaking conditions and a reliable setup at a synchrotron source, such as HZB BESSY II BL14.1 [2], preferably with some automation in initial data processing and refinement. A total hit rate greater than 10% was obtained, which will be compared to results from other screening methods. The resulting crystal structures will be discussed and provide an ideal basis for further lead development.

Drastic stabilization of parallel DNA hybridizations by a polylysine comb-type copolymer with hydrophilic graft chain.

Electrostatic interactions play a major role in protein-DNA interactions. As a model system of a cationic protein, herein we focused on a comb-type copolymer of a polycation backbone and dextran side chains, poly(L-lysine)-graft-dextran (PLL-g-Dex), which has been reported to form soluble interpolyelectrolyte complexes with DNA strands. We investigated the effects of PLL-g-Dex on the conformation and thermodynamics of DNA oligonucleotides forming various secondary structures. Thermodynamic analysis of the DNA structures showed that the parallel conformations involved in both DNA duplexes and triplexes were significantly and specifically stabilized by PLL-g-Dex. On the basis of thermodynamic parameters, it was further possible to design DNA switches that undergo structural transition responding to PLL-g-Dex from an antiparallel duplex to a parallel triplex even with mismatches in the third strand hybridization. These results suggest that polycationic molecules are able to induce structural polymorphism of DNA oligonucleotides, because of the conformation-selective stabilization effects.

Amyloid Fibril Formation by the Chain B Subunit of Monellin Occurs by a Nucleation-Dependent Polymerization Mechanism

Proteins possessing very different structures, or even no structure, form amyloid fibrils that are very similar in internal structure. This suggests that the mechanisms by which amyloid fibrils form might be very similar, irrespective of whether the fibrils are associated with disease or with normal cellular function, or even if they have no physiological importance. In this context, it is important to have a model protein system whose amyloid fibril formation is robust in its reproducibility, which can reveal the fundamentals of the amyloid fibril reaction that may be applicable to all proteins. In this study, the aggregation mechanism of amyloid fibril formation by chain B of the heterodimeric protein monellin has been elucidated in detail. It is shown that the aggregation reaction meets all the stringent kinetic criteria of a homogeneous nucleation-dependent polymerization mechanism, which is valid over a wide range of protein concentrations. Quantitative analyses of the kinetic data using one approach based on features of the entire kinetic curve, and another based on only the initial rate of aggregation, indicate that the thermodynamic nucleus is a dimer. Spherical oligomers are observed by atomic force microscopy to form transiently early during fibril formation but are off-pathway to the direct fibril formation pathway. It is shown that amyloid fibril formation can be prevented by the addition of chain A of monellin at early stages of chain B aggregation: the two free chains combine to form native monellin, which leads to the dissociation of early aggregates.

Binding Structure & Dynamics for Toxins Modifying the Gating Mechanism of Kv Channels

Voltage gated ion channels allow the flux of specific ions through the cell membrane to be controlled by the membrane potential, which makes them a critical component for electro-chemical signaling in the nervous system. The gating is regulated by the voltage sensor domains (VSD), in which the charged S4 helix moves to induce the conformational transition between open/closed states. This serves as an ideal model system for membrane proteins with multiple conformations, and if the gating can be modulated it would potentially enable a whole range of new drugs that could fine-tune the gating response. This is strikingly manifested in the case of tarantula spiders, which produce a potent venom that specifically target the VSD to causing dysfunctions in the neuronal and cardiac systems.Here, we report on molecular simulations of interactions between toxins and different Kv channels, in particular related to their influence of the gating transition. We have studied the binding to multiple intermediate states and effect of Stromatoxin (ScTx1) on gating transitions in both Kv2.1 and a mutant Shaker channel with enhanced toxin affinity. Results from toxin-protein docking show the toxin binding to S2 and S3 helices. An interaction pair is formed between E277 (Kv2.1 numbering) in the top-most part of S3 and either R4 or K22 on opposite sides of the toxin, resulting in two distinct poses in the binding site. In addition to docking, we have also explored multi-microsecond molecular dynamics simulations of toxins bound in both open and closed states.We propose a specific binding site for the toxin in the VSD of both channels, and predict that differences in residue interactions are responsible for selectively stabilizing S4 in either the open or closed state.

Effect of interactions with the chaperonin cavity on protein folding and misfolding.

Recent experimental and computational results have suggested that attractive interactions between a chaperonin and an enclosed substrate can have an important effect on the protein folding rate: it appears that folding may even be slower inside the cavity than under unconfined conditions, in contrast to what we would expect from excluded volume effects on the unfolded state. Here we examine systematically the dependence of the protein stability and folding rate on the strength of such attractive interactions between the chaperonin and substrate, by using molecular simulations of model protein systems in an idealised attractive cavity. Interestingly, we find a maximum in stability, and a rate which indeed slows down at high attraction strengths. We have developed a simple phenomenological model which can explain the variations in folding rate and stability due to differing effects on the free energies of the unfolded state, folded state, and transition state; changes in the diffusion coefficient along the folding coordinate are relatively small, at least for our simplified model. In order to investigate a possible role for these attractive interactions in folding, we have studied a recently developed model for misfolding in multidomain proteins. We find that, while encapsulation in repulsive cavities greatly increases the fraction of misfolded protein, sufficiently strong attractive protein-cavity interactions can strongly reduce the fraction of proteins reaching misfolded traps.

In Vivo Protein Footprinting for the Structural Analysis of Proteins in their Native Environment

Protein footprinting coupled with mass spectrometry has emerged in recent years as a valuable tool to study protein structure. Several footprinting techniques such as hydrogen deuterium exchange and oxidative labeling, have been successfully used to study many different protein systems. To date, these footprinting methods have been performed on relatively pure proteins in vitro. We have developed an in vivo oxidative labeling method to analyze proteins in their native environment. Oxidative labeling is advantageous for in vivo labeling owing to its irreversible nature. This allows for purification of the proteins of interest from the cell membrane. In vivo protein footprinting would be especially useful for membrane proteins and reduce the need for model membranes. Here we describe the development of in vivo oxidative labeling and its application to study ligand binding and conformational change of a model protein system.

Protein structure modeling for CASP10 by multiple layers of global optimization

In the template-based modeling (TBM) category of CASP10 experiment, we introduced a new protocol called protein modeling system (PMS) to generate accurate protein structures in terms of side-chains as well as backbone trace. In the new protocol, a global optimization algorithm, called conformational space annealing (CSA), is applied to the three layers of TBM procedure: multiple sequence-structure alignment, 3D chain building, and side-chain re-modeling. For 3D chain building, we developed a new energy function which includes new distance restraint terms of Lorentzian type (derived from multiple templates), and new energy terms that combine (physical) energy terms such as dynamic fragment assembly (DFA) energy, DFIRE statistical potential energy, hydrogen bonding term, etc. These physical energy terms are expected to guide the structure modeling especially for loop regions where no template structures are available. In addition, we developed a new quality assessment method based on random forest machine learning algorithm to screen templates, multiple alignments, and final models. For TBM targets of CASP10, we find that, due to the combination of three stages of CSA global optimizations and quality assessment, the modeling accuracy of PMS improves at each additional stage of the protocol. It is especially noteworthy that the side-chains of the final PMS models are far more accurate than the models in the intermediate steps.

Solid-State NMR Studies of Biomineralization Peptides and Proteins

Nature has evolved sophisticated strategies for engineering hard tissues through the interaction of proteins, and ultimately cells, with inorganic mineral phases. This process, called biomineralization, is how living organisms transform inorganic materials such as hydroxyapatite, calcite, and silica into highly intricate and organized structures. The remarkable material properties of shell, bone, and teeth come from the activities of proteins that function at the organic-inorganic interface. A better understanding of the biomolecular mechanisms used to promote or retard the formation of mineral-based structures could provide important design principles for the development of calcification inhibitors and promoters in orthopedics, cardiology, urology, and dentistry. With the knowledge of the structural basis for control of hard tissue growth by proteins, scientists could potentially develop materials using biomimetic principles with applications in catalysis, biosensors, electronic devices, and chromatographic separations, to name a few. Additionally, biomineralization also has potential applications in electronics, catalysis, magnetism, sensory devices, and mechanical design. Where man-made hard materials require the use of extreme temperatures, high pressure, and pH, biological organisms can accomplish these feats at ambient temperature and at physiological pH. Despite the fact that many researchers want to identify and control the structure of proteins at material and biomineral interfaces, there is a decided lack of molecular-level structure information available for proteins at biomaterial interfaces in general. In particular, this holds for mammalian proteins that directly control calcification processes in hard tissue. The most fundamental questions regarding the secondary and tertiary structures of proteins adsorbed to material surfaces, how proteins catalyze the formation of biomineral composites, or how proteins interact at biomaterial interfaces remain unanswered. This is largely due to a lack of methods capable of providing high-resolution structural information for proteins adsorbed to material surfaces under physiologically relevant conditions. In this Account, we highlight recent work that is providing insight into the structure and crystal recognition mechanisms of a salivary protein model system, as well as the structure and interactions of a peptide that catalyzes the formation of biosilica composites. To develop a better understanding of the structure and interactions of proteins in biomaterials, we have used solid-state NMR techniques to determine the molecular structure and dynamics of proteins and peptides adsorbed onto inorganic crystal surfaces and embedded within biomineral composites. This work adds to the understanding of the structure and crystal recognition mechanisms of an acidic human salivary phosphoprotein, statherin.

Infrared, Raman and INS studies of poly-ethylene oxide oligomers

In the present paper the experimental findings obtained by different spectroscopic techniques on water mixtures of a protein model system, i.e. PolyEthylene Oxide (PEO), are reported. By increasing the polymerization degree m, the D-LAM spectral contribution shows a behavior of the centre frequency and of linewidth which tends to reach a constant value. Also the intramolecular OH stretching contribution, by increasing m, shows a shift which tends to flatten for polymers with molecular weight higher than Mw=600. These results can be connected with the oligomer–polymer transition, at m=13. When in aqueous solutions the D-LAM frequency increases towards the value corresponding to that of the crystal phase; similarly one observes also a sharpening of the D-LAM spectral contribution. These evidences suggest the presence of a more ordered conformation of PEO in water in respect to the melt phase.

Brh2 and Rad51 promote telomere maintenance in Ustilago maydis, a new model system of DNA repair proteins at telomeres

Recent studies implicate a number of DNA repair proteins in mammalian telomere maintenance. However, because several key repair proteins in mammals are missing from the well-studied budding and fission yeast, their roles at telomeres cannot be modeled in standard fungi. In this report, we explored the dimorphic fungus Ustilago maydis as an alternative model for telomere research. This fungus, which belongs to the phylum Basidiomycota, has a telomere repeat unit that is identical to the mammalian repeat, as well as a constellation of DNA repair proteins that more closely mimic the mammalian collection. We showed that the two core components of homology-directed repair (HDR) in U. maydis, namely Brh2 and Rad51, both promote telomere maintenance in telomerase positive cells, just like in mammals. In addition, we found that Brh2 is localized to telomeres in vivo, suggesting that it acts directly at chromosome ends. We surveyed a series of mutants with DNA repair defects, and found many of them to have short telomeres. Our results indicate that factors involved in DNA repair are probably also needed for optimal telomere maintenance in U. maydis, and that this fungus is a useful alternative model system for telomere research.

On the role of surface composition and curvature on biointerface formation and colloidal stability of nanoparticles in a protein-rich model system

The need for a better understanding of nanoparticle–protein interactions and the mechanisms governing the resulting colloidal stability has been emphasised in recent years. In the present contribution, the short and long term colloidal stability of silica nanoparticles (SNPs) and silica–poly(ethylene glycol) nanohybrids (Sil–PEG) have been scrutinised in a protein model system. Well-defined silica nanoparticles are rapidly covered by bovine serum albumin (BSA) and form small clusters after 20min while large agglomerates are detected after 10h depending on both particle size and nanoparticle–protein ratio. Oppositely, Sil–PEG hybrids present suppressive protein adsorption and enhanced short and long term colloidal stability in protein solution. No critical agglomeration was found for either system in the absence of protein, proving that instability found for SNPs must arise as a consequence of protein adsorption and not to high ionic environment. Analysis of the small angle X-ray scattering (SAXS) structure factor indicates a short-range attractive potential between particles in the silica-BSA system, which is in good agreement with a protein bridging agglomeration mechanism. The results presented here point out the importance of the nanoparticle surface properties on the ability to adsorb proteins and how the induced or depressed adsorption may potentially drive the resulting colloidal stability.

Ultrasensitive and real-time detection of proteins in blood using a potentiometric carbon-nanotube aptasensor

Potentiometric sensing represents the preferred technique in many routine measurements of pH and ions. Unfortunately, the simplicity of the technique has not been exploited so far in high throughput biomolecular sensing. In this work, we demonstrate the capabilities of the hybrid functional material carbon nanotubes/aptamer for the creation of a new generation of nuclease-resistant aptasensors using the potentiometric transduction capabilities of single-walled carbon nanotubes in combination with the recognition capabilities of a protein-specific RNA aptamer. The aptasensor was used to detect and identify disease-related proteins at attomolar concentration values in a rapid and non-expensive way. The variable surface glycoprotein from African Trypanosomes was chosen as an ideal model system for a pathogenic exoantigen protein in a clinical sample. Variations in the electromotive force are achieved in real-time upon the direct addition of diluted real blood samples containing the target protein thus eliminating the need of preliminary matrix removal. This work would open the door to real-time diagnostic assays for a wide range of diseases, but also to the rapid molecular detection of several proteins in truly customizable protein biosensing platforms.

Nonionic Block Copolymers Assemble on the Surface of Protein Bionanoparticle

Efficient delivery of therapeutic proteins to a target site remains a challenge due to rapid clearance from the body. Here, we selected tobacco mosaic virus (TMV) as a model protein system to investigate the interactions between the protein and a nonionic block copolymer as a possible protecting agent for the protein. By varying the temperature, we were able to obtain core-shell structures based on hydrophobic interactions among PO blocks and noncovalent interactions between TMV and EO blocks. The protein-polymer interactions were characterized by dynamic light scattering and isothermal titration calorimetry. This study establishes principles for the possible design of clinically useful protein delivery systems.

Toward molecular models of proton pumping: Challenges, methods and relevant applications

Motivated by several long-lasting mechanistic questions for biomolecular proton pumps, we have engaged in developing hybrid quantum mechanical/molecular mechanical (QM/MM) methods that allow an efficient and reliable description of long-range proton transport in transmembrane proteins. In this review, we briefly discuss several relevant issues: the need to develop a “multi-scale” generalized solvent boundary potential (GSBP) for the analysis of chemical events in large trans-membrane proteins, approaches to validate such a protocol, and the importance of improving the flexibility of QM/MM Hamiltonian. Several recent studies of model and realistic protein systems are also discussed to help put the discussions into context. Collectively, these studies suggest that the QM/MM-GSBP framework based on an approximate density functional theory (SCC-DFTB) as QM holds the promise to strike the proper balance between computational efficiency, accuracy and generality. With additional improvements in the methodology and recent developments by others, especially powerful sampling techniques, this “multi-scale” framework will be able to help unlock the secrets of proton pumps and other biomolecular machines.

A Desolvation Model for Trifluoroethanol-Induced Aggregation of Enhanced Green Fluorescent Protein

Studies of amyloid disease-associated proteins in aqueous solutions containing 2,2,2-trifluoroethanol (TFE) have shown that the formation of structural intermediates is often correlated with enhanced protein aggregation. Here, enhanced green fluorescent protein (EGFP) is used as a model protein system to investigate the causal relationship between TFE-induced structural transitions and aggregation. Using circular dichroism spectroscopy, light scattering measurements, and transmission electron microscopy imaging, we demonstrate that population of a partially α-helical, monomeric intermediate is roughly correlated with the growth of β-sheet-rich, flexible fibrils for acid-denatured EGFP. By fitting our circular dichroism data to a model in which TFE-water mixtures are assumed to be ideal solutions, we show that increasing entropic costs of protein solvation in TFE-water mixtures may both cause the population of the intermediate state and increase aggregate production. Tertiary structure and electrostatic repulsion also impede aggregation. We conclude that initiation of EGFP aggregation in TFE likely involves overcoming of multiple protective factors, rather than stabilization of aggregation-prone structural elements.