Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

Micelles,Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.     

Laser‐Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass‐Spectrometry‐Based Protein Footprinting

Described is a novel, laser‐initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. ^.CF₃ reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to ^.OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chemistry and structural biology and biotechnology. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility.     

Future directions of structural mass spectrometry using hydroxyl radical footprinting

Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein–protein and protein–DNA interactions. Using synchrotron radiolysis, exposure of proteins to a ‘white’ X‐ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time‐resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium‐dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time‐resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)‐based method can be utilized for quantification of oxidized species, improving the signal‐to‐noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis‐driven structural mass spectrometry experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

Membrane-bound alpha-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles

We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (alphaS) bound to small unilamellar phospholipid vesicles, rodlike SDS micelles, or lipid bicelles. By measuring distances as long as approximately 7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that alphaS does not retain a "hairpin" structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the interhelix linker. While the possibility of local helix discontinuities in the structure of membrane-bound alphaS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.     

Self‐Assembled Polyprodrug Amphiphile for Subcutaneous Xenograft Tumor Inhibition with Prolonged Acting Time In Vivo

Polymeric drug delivery system termed as “polyprodrug amphiphile” poly(2‐methylacryloyloxyethyl phosphorylcholine)‐b‐poly(10‐hydroxy‐camptothecin methacrylate (pMPC‐b‐pHCPT) is developed for the prolonged‐acting cancer therapy. It is obtained by two‐step reversible addition–fragmentation chain transfer polymerization of zwitterionic monomer MPC and an esterase‐responsive polymerizable prodrug methacrylic anhydride–CPT, respectively. This diblock polymer is composed of both antifouling (pMPC) and bioactive (pHCPT) segments and the drug is designed as a building block to construct the polymer skeleton directly. Due to its distinct amphiphilicity, the polymer can self‐assemble into micelles with different dynamic sizes by facilely tuning the ratio of MPC/HCPT under physiological conditions. The outer pMPC shell is superhydrophilic to form dense hydrate layer preventing the nanosystem from unwanted nonspecific protein adsorption, which is the main lead cause of the rapid clearance of nanoparticles in vivo, thus facilitating the accumulation of drugs in tumor sites via enhanced permeability and retention effect. The configuration of the polyprodrug amphiphile is confirmed by several measurements. The resistance to albumin adsorption, prolonged plasma retention time, accumulation in tumor sites, and anticancer activity of the micelles is also investigated in vitro and in vivo. This novel amphiphile can be expected as a promising agent for the passive targeted prolonged‐acting cancer therapy.     

Reduction‐Triggered Transformation of Disulfide‐Containing Micelles at Chemically Tunable Rates

A dilemma exists between the circulation stability and cargo release/mass diffusion at desired sites when designing delivery nanocarriers and in vivo nanoreactors. Reported herein are disulfide‐crosslinked (DCL) micelles exhibiting reduction‐triggered switching of crosslinking modules and synchronized hydrophobic‐to‐hydrophilic transition. Tumor cell targeted DCL micelles undergo cytoplasmic milieu triggered disulfide cleavage and self‐immolative decaging reactions at chemically adjustable rates, generating primary amine moieties. Extensive amidation reactions with neighboring ester moieties then occur because of the high local concentration and suppression of the apparent amine pK_a value within the hydrophobic cores, thus leading to the transformation of crosslinking modules and formation of tracelessly crosslinked (TCL) micelles, with hydrophilic cores, inside live cells. We further integrate this design principle with theranostic nanocarriers for selective intracellular drug transport guided by enhanced magnetic resonance (MR) imaging performance.     

Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side‐chain dynamics of the α‐helical sensory rhodopsin II and the β‐barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl‐bearing side‐chain motion that is largely independent of membrane mimetic. The methyl‐bearing side chains of both proteins are, on average, more dynamic in the ps–ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane‐protein functions, including ligand binding, allostery, and signaling.     

Temperature‐Dependent Atomic Models of Detergent Micelles Refined against Small‐Angle X‐Ray Scattering Data

Surfactants have found a wide range of industrial and scientific applications. In particular, detergent micelles are used as lipid membrane mimics to solubilize membrane proteins for functional and structural characterization. However, an atomic‐level understanding of surfactants remains limited because many experiments provide only low‐resolution structural information on surfactant aggregates. In this work, small‐angle X‐ray scattering is combined with molecular dynamics simulations to derive fully atomic models of two maltoside micelles at temperatures between 10 °C and 70 °C. The micelles take the shape of general tri‐axial ellipsoids and decrease in size and aggregation number with increasing temperature. Density profiles of hydrophobic groups and water along the three principal axes reveal that the minor micelle axis closely mimics lipid membranes. The results suggest that coupling atomic simulations with low‐resolution data allows the structural characterization of surfactant aggregates.     

Bilayer in small bicelles revealed by lipid-protein interactions using NMR spectroscopy

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and small bicelles of dihexanoyl phosphatidylcholine (DHPC) and dimyristoyl phosphatidylcholine (DMPC) give insights into protein-lipid interactions. Intermolecular NOEs between hydrophobic tails of lipid and protein in the bicelles cover the surface area of OmpX forming a continuous cylindric jacket of approximately 2.7 nm in height. These NOEs originate only from DMPC molecules, and no NOEs from DHPC are observed. Further, these NOEs are mainly from methylene groups of the hydrophobic tails of DMPC, and only a handful of NOEs arise from methyl groups of the hydrophobic tails. The observed contacts indicate that the hydrophobic tails of DMPC are oriented parallel to the surface of OmpX and thus DMPC molecules form a bilayer in the vicinity of the protein. Thus, a bilayer exists in the small bicelles not only in the absence of but also in the presence of a membrane protein. In addition, the number of NOEs between the polar head groups of lipid molecules and protein is increased in the bicelles compared with those in micelles. This observation may be due to the closely packed head groups of the bilayer. Moreover, irregularity of hydrophobic interactions in the middle of the bilayer environment was observed. This observation together with the interactions between polar head groups and proteins gives a possible rationale for structural and functional differences of membrane proteins solubilized in micelles and in bilayer systems and hints at structural differences between protein-free and protein-loaded bilayers.     

Self‐Assembly Behaviors of a Penta‐Phenylene Maltoside and Its Application for Membrane Protein Study

We prepared an amphiphile with a penta‐phenylene lipophilic group and a branched trimaltoside head group. This new agent, designated penta‐phenylene maltoside (PPM), showed a marked tendency to self‐assembly into micelles via strong aromatic–aromatic interactions in aqueous media, as evidenced by ¹H NMR spectroscopy and fluorescence studies. When utilized for membrane protein studies, this new agent was superior to DDM, a gold standard conventional detergent, in stabilizing multiple proteins long term. The ability of this agent to form aromatic–aromatic interactions is likely responsible for enhanced protein stabilization when associated with a target membrane protein.     

Biocompatibility evaluation of self‐assembled micelles prepared from poly(lactide‐co‐glycolide)‐poly(ethylene glycol) diblock copolymers

In this work, a series of PLGA‐PEG diblock copolymers were synthesized by ring‐opening polymerization of L‐lactide and glycolide using mPEG as macroinitiator and stannous octoate as catalyst. Spherical micelles were obtained from the various copolymers by using co‐solvent evaporation method. The biocompatibility of micelles was evaluated with the aim of assessing their potential in the development of drug delivery systems. Various aspects of biocompatibility were considered, including MTT assay, agar diffusion test, release of cytokines, hemolytic test, dynamic clotting time, protein adsorption in vitro, and zebrafish embryonic compatibility in vivo. The combined results revealed that the micelles present good cytocompatibility and hemocompatibility in vitro. Moreover, the cumulative effects of micelles throughout embryos developing stages have no toxicity in vivo. It is thus concluded that micelles prepared from PLGA‐PEG copolymers present good biocompatibility as potential drug carrier.     

Targeted Antithrombotic Protein Micelles

Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site‐specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one‐pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single‐chain antibody (scFv), which binds to the ligand‐induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.     

Striking Improvement in Peroxidase Activity of Cytochrome c by Modulating Hydrophobicity of Surface‐Functionalized Gold Nanoparticles within Cationic Reverse Micelles

This work demonstrates a remarkable enhancement in the peroxidase activity of mitochondrial membrane protein cytochrome c (cyt c) by perturbing its tertiary structure in the presence of surface‐functionalised gold nanoparticles (GNPs) within cetyltrimethylammonium bromide (CTAB) reverse micelles. The loss in the tertiary structure of cyt c exposes its heme moiety (which is buried inside in the native globular form), which provides greater substrate (pyrogallol and H₂O₂) accessibility to the reactive heme residue. The surfactant shell of the CTAB reverse micelle in the presence of co‐surfactant (n‐hexanol) exerted higher crowding effects on the interfacially bound cyt c than similar anionic systems. The congested interface led to protein unfolding, which resulted in a 56‐fold higher peroxidase activity of cyt c than that in water. Further perturbation in the protein’s structure was achieved by doping amphiphile‐capped GNPs with varying hydrophobicities in the water pool of the reverse micelles. The hydrophobic moiety on the surface of the GNPs was directed towards the interfacial region, which induced major steric strain at the interface. Consequently, interaction of the protein with the hydrophobic domain of the amphiphile further disrupted its tertiary structure, which led to better opening up of the heme residue and, thereby, superior activity of the cyt c. The cyt c activity in the reverse micelles proportionately enhanced with an increase in the hydrophobicity of the GNP‐capping amphiphiles. A rigid cholesterol moiety as the hydrophobic end group of the GNP strikingly improved the cyt c activity by up to 200‐fold relative to that found in aqueous buffer. Fluorescence studies with both a tryptophan residue (Trp59) of the native protein and the sodium salt of fluorescein delineated the crucial role of the hydrophobicity of the GNP‐capping amphiphiles in improving the peroxidase activity of cyt c by unfolding its tertiary structure within the reverse micelles.     

A New Colorimetric Platform for Protein Detection Based on Recognition‐Induced Cascade of Polymeric Nanoparticles Disassembly

Despite great progress, it is still of high interest to explore new homogeneous assays for simple, visual, and selective protein detection. Herein, one new colorimetric sensor has been developed for visual detection of protein by using polymeric micelles as a sensing scaffold and the molecular recognition between protein and the ligand on the surface of the polymeric micelles as the driving force to trigger the readout of the detection signal. The polymeric micelles formed via the self‐assembly of the amphiphilic block polymer biotin‐labeled poly(ethylene glycol)‐block‐poly(3‐acryl aminophenylboronic acid) are endowed with colorful feature by incorporation of alizarin red S (ARS) into the hydrophobic core. Based on the response to streptavidin recognition, these micelles are further disintegrated through the competitive binding of α‐cyclodextrin with boronic acid for disassociation of ARS, which achieves orange–yellow to pink–purple transition in 2 h. This work will open the way to develop one new mix‐and‐measure, visual, and homogeneous assay.     

Prediction of membrane spanning segments and topology in β‐barrel membrane proteins at better accuracy

Prediction of membrane spanning segments in β‐barrel outer membrane proteins (OMP) and their topology is an important problem in structural and functional genomics. In this work, we propose a method based on radial basis networks for predicting the number of β‐strands in OMPs and identifying their membrane spanning segments. Our method showed a leave‐one‐out cross validation accuracy of 96% in a set of 28 OMPs, which have the range of 8–22 β‐strand segments. The β‐strand segments in OMPs and the residues in membrane spanning segments are correctly predicted with the accuracy of 96% and 87%, respectively. We have developed a web server, TMBETAPRED‐RBF for predicting the transmembrane β‐strands from amino acid sequence and it is available at http://rbf.bioinfo.tw/～sachen/tmrbf.html . We suggest that our method could be an effective tool for predicting the membrane spanning regions and topology of β‐barrel membrane proteins. © 2009 Wiley Periodicals, Inc. J Comput Chem 2010     

Modulation of Substrate Specificity upon Modification of Cholesterol Oxidase from Brevibacterium Sterolicum by Hydrogen Peroxide: Evidence for a Peripheral Site

Evidence for a peripheral site that recognizes micelles where cholesterol is inserted, is presented for cholesterol oxidase from Brevibacterium sterolicum (CHOD). The Ω loop part of this site contains methionine residues. Their oxidation is reflected in a different selectivity to the micelles. When CHOD is oxidized with hydrogen peroxide, preferential introduction of two oxygen atoms by hydrogen peroxide to the enzyme is observed by the electrospray mass spectrometry. The modified enzyme does not oxidize cholesterol inserted in H‐Triton X‐100/sodium cholate mixed micelles. However, cholesterol solubilized in microemulsions, in biphasic media or inserted in hydroxypropyl‐β‐cyclodextrin is oxidized by the modified enzyme. This modified enzyme oxidizes pregn‐5‐en‐3β‐ol solubilized in mixed micelles, and soluble steroids, such as 3β‐hydroxyandrost‐5‐en‐17β‐carboxylic acid or 3β‐hydroxyandrost‐5‐en‐17‐one. So the modification does not occur at the active site, but at the peripheral site (methionine 81), abolishing the recognition of the micelles and thus inactivating the enzyme specifically towards cholesterol inserted in the micelles.     

DNA‐Based Micelles: Synthesis,Micellar Properties and Size‐Dependent Cell Permeability

Functional nanomaterials based on molecular self‐assembly hold great promise for applications in biomedicine and biotechnology. However, their efficacy could be a problem and can be improved by precisely controlling the size, structure, and functions. This would require a molecular engineering design capable of producing monodispersed functional materials characterized by beneficial changes in size, shape, and chemical structure. To address this challenge, we have designed and constructed a series of amphiphilic oligonucleotide molecules. In aqueous solutions, the amphiphilic oligonucleotide molecules, consisting of a hydrophilic oligonucleotide covalently linked to hydrophobic diacyllipid tails, spontaneously self‐assemble into monodispersed, three‐dimensional micellar nanostructures with a lipid core and a DNA corona. These hierarchical architectures are results of intermolecular hydrophobic interactions. Experimental testing further showed that these types of micelles have excellent thermal stability and their size can be fine‐tuned by changing the length of the DNA sequence. Moreover, in the micelle system, the molecular recognition properties of DNA are intact, thus, our DNA micelles can hybridize with complimentary sequences while retaining their structural integrity. Importantly, when interacting with cell membranes, the highly charged DNA micelles are able to disintegrate themselves and insert into the cell membrane, completing the process of internalization by endocytosis. Interestingly, the fluorescence was found accumulated in confined regions of cytosole. Finally, we show that the kinetics of this internalization process is size‐dependent. Therefore, cell permeability, combined with small sizes and natural nontoxicity are all excellent features that make our DNA–micelles highly suitable for a variety of applications in nanobiotechnology, cell biology, and drug delivery systems.     

Molecular Alliance—From Orthosteric and Allosteric Ligands to Dualsteric/Bitopic Agonists at G Protein Coupled Receptors

Cell‐membrane‐spanning G protein coupled receptors (GPCRs) belong to the most important therapeutic target structures. Endogenous transmitters bind from the outer side of the membrane to the “orthosteric” binding site either deep in the binding pocket or at the extracellular N‐terminal end of the receptor protein. Exogenous modulators that utilize a different, “allosteric”, binding site unveil a pathway to receptor subtype‐selectivity. However, receptor activation through the orthosteric area is often more powerful. Recently there has been evidence that orthosteric/allosteric, in other words “dualsteric”, hybrid compounds unite subtype selectivity and receptor activation. These “bitopic” modulators channelreceptor activation and subsequent intracellular signaling into a subset of possible routes. This concept offers access to GPCR modulators with an unprecedented receptor‐subtype and signaling selectivity profile and, as a consequence, to drugs with fewer side effects.     

Solubilization of Membrane Proteins into Functional Lipid‐Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer

Once removed from their natural environment, membrane proteins depend on membrane‐mimetic systems to retain their native structures and functions. To this end, lipid‐bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane‐protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl‐chain order, does not interfere with optical spectroscopy in the far‐UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations.