Evan K. Brooks

Structure and dynamics of a conformationally constrained nitroxide side chain and applications in EPR spectroscopy

A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed spin labeling. Although the internal motion of R1 and the number of preferred rotamers are limited, translating interspin distance measurements and spatial orientation information into structural constraints is challenging. Here, we introduce a highly constrained nitroxide side chain designated RX as an alternative to R1 for these applications. RX is formed by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i + 4 in an α-helix, at i and i + 2 in a β-strand, or with cysteine residues in adjacent strands in a β-sheet. Analysis of EPR spectra, a crystal structure of RX in T4 lysozyme, and pulsed electron-electron double resonance (ELDOR) spectroscopy on an immobilized protein containing RX all reveal a highly constrained internal motion of the side chain. Consistent with the constrained geometry, interspin distance distributions between pairs of RX side chains are narrower than those from analogous R1 pairs. As an important consequence of the constrained internal motion of RX, spectral diffusion detected with ELDOR reveals microsecond internal motions of the protein. Collectively, the data suggest that the RX side chain will be useful for distance mapping by EPR spectroscopy, determining spatial orientation of helical segments in oriented specimens, and measuring structural fluctuations on the microsecond time scale.

Orthogonal spin labeling and Gd(III)-nitroxide distance measurements on bacteriophage T4-lysozyme.

We present the first example of chemoselective site-specific spin labeling of a monomeric protein with two spectroscopically orthogonal spin labels: a gadolinium(III) chelate complex and a nitroxide radical. A detailed analysis of the performance of two commercially available Gd(III) ligands in the Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) experiment is reported. A modification of the flip angle of the pump pulse in the Gd(III)-nitroxide DEER experiment is proposed to optimize sensitivity.

Synthesis and characterization of nanoscale biomimetic polymer vesicles and polymer membranes for bioelectronic applications

An amphiphilic ABA triblock copolymer was synthesized using poly(2-ethyl-2-oxazoline) (PEtOz) as hydrophilic block [A] and poly(dimethylsiloxane) (PDMS) as hydrophobic block [B]. The cationic ring-opening polymerization of 2-ethyl-2-oxazoline was initiated by benzyl chloride in the presence of NaI. PEtOz–PDMS–PEtOz was characterized in aqueous solution using transmission electron microscopy (TEM). The block copolymers formed vesicles with a [B] block hydrophobic component thickness of 4 nm, which is thin enough for successful reconstitution of proteins. The mean diameters of the vesicles were measured to be in the range of 150–250 nm, with a narrow distribution. The electrochemical properties of planar PEtOz–PDMS–PEtOz membrane films spread across a Teflon aperture were investigated by electrochemical impedance spectroscopy (EIS). The impedance data showed an increase of planar membrane capacitance (CMEM) from 2.58 × 10−7 to 2.71 × 10−7 F cm−2 and a decrease of membrane resistance (RMEM) from 12 to 10.8 Ω cm2. The increase of CMEM over time in buffer solution can be explained by an increase of the dielectric constant as a result of membrane electrolyte incorporation and/or by the formation and growth of defect in the free-standing films. In contrast to the formation of thin-walled vesicles, the spread triblock copolymer formed a free-standing membrane with a 9 nm thickness. Based on the two possible conformations (bridge midblock conformation and loop midblock conformation) that the triblock copolymer can have, we can conclude that PEtOz–PDMS–PEtOz formed 4 nm thick polymer vesicles with intercalated loop midblock structure in aqueous solution, while 9 nm thick free-standing polymer films with bilayer loop midblock conformation or with bridge midblock conformation were formed by aperture spreading.

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

Significance Excited states of proteins play functional roles, but their low population and conformational flexibility pose a challenge for characterization by most spectroscopic techniques. Here, this challenge is met by combining high hydrostatic pressure, which reversibly populates excited states, and site-directed spin labeling with double electron–electron resonance (DEER) spectroscopy, which resolves distinct conformational substates of proteins by measuring distances between spin-labeled pairs. We present a method for trapping high-pressure equilibria of proteins by rapid freezing under pressure, followed by depressurization and acquisition of DEER data at atmospheric pressure. The methodology is applied to myoglobin, revealing unique information on the length scale of helical fluctuations in the pressure-populated as compared with the pH-populated molten globule states of the apo-protein. The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron–electron resonance (DEER) provides long-range (20–80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0–3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter.

Fabrication of biomolecule-copolymer hybrid nanovesicles as energy conversion systems

This work demonstrates the integration of the energy-transducing proteins bacteriorhodopsin (BR) from Halobacterium halobium and cytochrome c oxidase (COX) from Rhodobacter sphaeroides into block copolymeric vesicles towards the demonstration of coupled protein functionality. An ABA triblock copolymer-based biomimetic membrane possessing UV-curable acrylate endgroups was synthesized to serve as a robust matrix for protein reconstitution. BR-functionalized polymers were shown to generate light-driven transmembrane pH gradients while pH gradient-induced electron release was observed from COX-functionalized polymers. Cooperative behaviour observed from composite membrane functionalized by both proteins revealed the generation of microamp-range currents with no applied voltage. As such, it has been shown that the fruition of technologies based upon bio-functionalizing abiotic materials may contribute to the realization of high power density devices inspired by nature.

Engineering Visual Arrestin-1 with Special Functional Characteristics

Background: Arrestin-1 with enhanced binding to unphosphorylated active rhodopsin (Rh*) has therapeutic potential. Results: Manipulation of the rhodopsin binding surface of arrestin-1 greatly increases its binding to Rh*. Conclusion: Stable arrestin-1 with high binding to Rh* can be engineered with and without the ability to self-associate. Significance: The affinity of arrestin-1 for Rh* and its propensity to oligomerize can be independently changed by targeted mutagenesis. Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations.

Hybrid protein-polymer biomimetic membranes

Protein-functionalized polymers retain dramatically increased stability over lipid membranes and the unique ability to be deposited on solid substrates in the ABA complex. Furthermore, since these polymers can mimic hydrophilic/hydrophobic biological environments in a single molecular chain, direct adsorption of protein-functionalized biomembrane films is enabled, which is a significant advantage over conventional lipid systems. Following the demonstration of protein mutagenesis and nanoscale biomimetic membrane fabrication, monolayer arrays of pore proteins have been deposited onto silicon wafers for applications in sensing nanomolecules such as conjugated quantum dots and colloidal gold beads. Furthermore, we have characterized monolayer surface properties of custom tailored polymers with varied block length for biomimetic membrane applications, as well as developed a multiwell microelectromechanical-system-based membrane testing platform for enhanced versatility in film deposition. We have successfully demonstrated the reconstitution of a genetically engineered OmpF porin in block copolymer-based biomembranes, fabrication of large-area hybrid protein-polymer Langmuir-Blodgett films, as well as protein insertion via macromolecule detection using protein-polymer active materials with the goal of buildup toward a multicomponent microsystem while preserving inherent molecular function.

Hybrid protein/polymer biomimetic membranes

Protein-functionalized polymers retain dramatically increased stability over lipid membranes and the unique ability to be deposited on solid substrates in the ABA complex. Monolayer arrays of pore proteins have been deposited onto silicon wafers for applications in sensing nanomolecules such as conjugated quantum dots and colloidal gold beads. We have successfully demonstrated the reconstitution of the OmpF porin in triblock copolymer membranes, fabrication of large-area hybrid Langmuir-Blodgett Films, as well as protein insertion via analyte detection using protein/polymer active materials with the goal of buildup towards a multicomponent microsystem while preserving inherent molecular function.

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant.

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization.

A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu(2+) bound to an engineered Cu(2+) binding site, enhanced T1 relaxation of TAM should enable measurement of interspin distances up to 50Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins.