Peter F. Flynn

Reverse Micelle Encapsulation as a Model for Intracellular Crowding

Reverse micelles are discrete nanoscale particles composed of a water core surrounded by surfactant. The amount of water within the core of reverse micelles can be easily manipulated to directly affect the size of the reverse micelle particle. The water loading capacity of reverse micelles varies with temperature, and water can be shed if reverse micelles are exposed to low temperatures. The use of water shedding from the reverse micelle provides precise and comprehensive control over the amount of water available to solvate host molecules. Proteins encapsulated within reverse micelles can be studied to determine the effects of confinement and excluded volume. The data presented here provide an important bridge between commonly employed dilute in vitro studies and studies of the effects of a crowded environment, as found in vivo. Ubiquitin was encapsulated within bis(2-ethylhexyl) sodium sulfosuccinate AOT reverse micelles under various degrees of confinement and was compared with an analogously reconstituted sample of ubiquitin in the commonly used molecular crowding agent bovine serum albumin. The effects of encapsulation were monitored using chemical shift perturbation analysis of the amide (1)H and (15)N resonances. The results also reconcile alternative interpretations of protein cold denaturation within reverse micelles.

Preparation, characterization, and NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

Encapsulating a protein in a reverse micelle and dissolving it in a low-viscosity solvent can lower the rotational correlation time of a protein and thereby provides a novel strategy for studying proteins in a variety of contexts. The preparation of the sample is a key element in this approach and is guided by a number of competing parameters. Here we examine the applicability of several strategies for the preparation and characterization of encapsulated proteins dissolved in low viscosity fluids that are suitable for high performance NMR spectroscopy. Ubiquitin is used as a model system to explore various issues such as the homogeneity of the encapsulation, characterization of the hydrodynamic performance of reverse micelles containing protein molecules, and the effective pH of the water environment of the reverse micelle.

Intrinsic Domain and Loop Dynamics Commensurate with Catalytic Turnover in an Induced-Fit Enzyme

Arginine kinase catalyzes reversible phosphoryl transfer between ATP and arginine, buffering cellular ATP concentrations. Structures of substrate-free and -bound enzyme have highlighted a range of conformational changes thought to occur during the catalytic cycle. Here, NMR is used to characterize the intrinsic backbone dynamics over multiple timescales. Relaxation dispersion indicates rigid-body motion of the N-terminal domain and flexible dynamics in the I182-G209 loop, both at millisecond rates commensurate with k(cat), implying that either might be rate limiting upon catalysis. Lipari-Szabo analysis indicates backbone flexibility on the nanosecond timescale in the V308-V322 loop, while the rest of the enzyme is more rigid in this timescale. Thus, intrinsic dynamics are most prominent in regions that have been independently implicated in conformational changes. Substrate-free enzyme may sample an ensemble of different conformations, of which a subset is selected upon substrate binding, with critical active site residues appropriately configured for binding and catalysis.

Preparation of encapsulated proteins dissolved in low viscosity fluids

The majority of proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. One potential approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques is to encapsulate them in a reverse micelle which is dissolved in a low viscosity fluid. Unfortunately, promising low viscosity fluids such as the short chain alkanes, supercritical carbon dioxide, and various halocarbon refrigerants all require the application of significant pressure to be kept liquefied at room temperature. Here we describe the design and use of a simple cost effective NMR tube suitable for the preparation of solutions of proteins encapsulated in reverse micelles dissolved in such fluids.

Use of reverse micelles in membrane protein structural biology

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques.

NMR Studies of Encapsulated Macromolecules

Encapsulation of proteins with reverse micelles has recently been identified as an important new biological NMR application. Encapsulation involves transfer of a hydrated protein into the interior chamber formed within an inverted shell of surfactant (usually dioctyl sulfosuccinate), forming a particle that is dissolved in a low-viscosity solvent, most commonly a short chain alkane. Experimental evidence has demonstrated that macromolecules of significant size and complexity can be encapsulated, and that under appropriate conditions encapsulated molecules retain native structure and biological activity. The tumbling rate of an encapsulated protein is roughly proportional to the bulk viscosity of the solvent, and by selecting an appropriately low viscosity liquid the tumbling rate of an encapsulated molecule may be significantly over that that measured for the free protein in aqueous solution. Such samples may exhibit spectroscopic properties that are superior to those of the free molecules in aqueous solution, and encapsulation thus promises to provide an important enhancement to the resolution and sensitivity of solution NMR experiments. In addition to thebenefits associated with increases in the rate of tumbling, encapsulation has also been shown to be an important biophysical technique that can be used to investigate the influence of environment on proteins. The function of proteins depends not only on the physiochemical attributes of the molecules themselves but also on the local environment in which the molecules are active. Reverse micelle based encapsulation is capable of producing novel environments that serve as a platform for studying the influence of confinement, hydration, ionic strength, and temperature in limits that are beyond the scope of other experimental approaches.

Influence of induced fit in the interaction of Bacillus subtilis trp RNA-binding attenuator protein and its RNA antiterminator target oligomer

In the presence of excess tryptophan, tryptophan‐activated TRAP (trp RNA‐binding attenuator protein) binds to a specific target in the trp‐leader transcript, which induces the formation of a transcription terminator and transcription halts in the leader region. In the absence of tryptophan, TRAP does not bind RNA, an antiterminator forms, and the operon is expressed. Although the ternary complex involving TRAP (Bacillus stearothermophilus), tryptophan, and the RNA target has recently been crystallized, efforts to obtain structural data for the apo‐form of TRAP (in any species) have not been successful. We have used multidimensional/multinuclear nuclear magnetic resonance (NMR) spectroscopy to probe the structure–function relationship in the TRAP‐activated system, and have obtained high‐resolution multidimensional/multinuclear NMR spectra of TRAP in all three of its functional states: tryptophan‐free or apo‐TRAP, tryptophan‐activated TRAP, and tryptophan‐activated TRAP‐RNA ternary complex. Chemical shift perturbation analysis of the NMR data clarifies the interpretation of results obtained from previous crystal studies. Results presented herein demonstrate that tryptophan binding induces an essential structural change in TRAP that supports high‐affinity binding of the RNA target oligonucleotide. Proteins 2002;49:432–438.

NMR Spectroscopy of Encapsulated Proteins Dissolved in Low Viscosity Fluids

The challenges to solution NMR spectroscopy originating from the slow tumbling of large proteins are reviewed and a novel solution based on a hydrodynamic approach is described. This solution is based on the effective decrease in tumbling time brought about by the encapsulation of the protein of interest in a protective reverse micelle assembly and its subsequent dissolution in a low viscosity fluid. The physical basis of the method is reviewed and a variety of technical issues are discussed. Recent advances that appear to validate the approach as a tool for structuralbiology are presented. Future applications and developments are also described.

Assignment of 1H, 13C, and 15N resonances of the RNA binding protein Snu13p from Saccharomyces cerevisiae

Snu13p is a highly conserved RNA binding protein from Saccharomyces cerevisiae required for both eukaryotic pre-mRNA splicing and pre-rRNA processing. The 1H, 13C, and 15N assignments were determined from multidimensional, multinuclear NMR experiments conducted at 25°C.

Measuring and modeling highly accurate 15N chemical shift tensors in a peptide.

NMR studies measuring chemical shift tensors are increasingly being employed to assign structure in difficult-to-crystallize solids. For small organic molecules, such studies usually focus on 13 C sites, but proteins and peptides are more commonly described using 15 N amide sites. An important and often neglected consideration when measuring shift tensors is the evaluation of their accuracy against benchmark standards, where available. Here we measure 15 N tensors in the dipeptide glycylglycine at natural abundance using the slow-spinning FIREMAT method with SPINAL-64 decoupling. The accuracy of these 15 N tensors is evaluated by comparing to benchmark single crystal NMR 15 N measurements and found to be statistically indistinguishable. These FIREMAT experimental results are further used to evaluate the accuracy of theoretical predictions of tensors from four different density functional theory (DFT) methods that include lattice effects. The best theoretical approach provides a root mean square (rms) difference of ±3.9 ppm and is obtained from a fragment-based method and the PBE0 density functional.