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Silica nanoparticles (SiNPs) are important nano-sized, solid-state carriers/hosts to load, store, and deliver biological or pharmaceutical cargoes. They are also good potential solid supports to immobilize proteins for fundamental protein structure and dynamics studies. However, precaution is necessary when using SiNPs in these areas because adsorption might alter the activity of the cargoes, especially when enzymes are loaded. Therefore, it becomes important to understand the structural basis of the cargo enzyme activity changes, if there is any. The high complexity and dynamics of the nano-bio interface present many challenges. Reported here is a comprehensive study of the structure, dynamics, and activity of a model enzyme, T4 lysozyme, upon adsorption to a few surface-modified SiNPs using several experimental techniques. Not surprisingly, a significant activity loss on each studied SiNP was found. The structural basis of the activity loss was identified based on results from a unique technique, the Electron Paramagnetic Resonance (EPR) spectroscopy, which probes structural information regardless of the complexity. Several docking models of the enzyme on SiNPs with different surfaces, at different enzyme-to-SiNP ratios are proposed. Interestingly, we found that the adsorbed enzyme can be desorbed via pH adjustment, which highlighted the potential to use SiNPs for enzyme/protein delivery or storage due to the high capacity. In order to use SiNPs as enzyme hosts, minimizing the enzymatic activity loss upon adsorption is needed. Lastly, the work outlined here demonstrate the use of EPR in probing structural information on the complex (inorganic)nano-bio interface.

The squeeze film damper (SFD) is a damping device that has been widely used in rotating machines. The SFD can effectively suppress rotor vibration and reduce transmitted forces. However, it would cause complex motions of the rotor system under specific operating conditions. This paper investigates the support structure of a high-pressure stage simulated rotor in an aircraft engine. A dynamic model of the SFD considering the effects of inertia and cavitation is established to investigate the influence of structural parameters on the stiffness and damping coefficient of the damper. Subsequently, a finite element model of the Jeffcott rotor–SFD system is developed to explore the effects of the structure parameters of the SFD on the critical speed, mode shapes, and other dynamic characteristics of the Jeffcott rotor–SFD system. Experimental tests are conducted to validate and refine the numerical calculations regarding the dynamic characteristics of the rotor–damper system. The results indicate that there is consistency between numerical simulations and experimental results. Within a certain range, enlarging the width of the damper can significantly increase the magnitude of the pressure distribution of the oil film. However, when the pressure amplitude increases to the saturation vapor pressure of the lubricating oil, the cavitation effect begins to manifest. As the width of the damper increases, both the stiffness coefficient and damping coefficient of the SFD are enhanced. Consequently, the vibration reduction effectiveness of the SFD is significantly improved. [ABSTRACT FROM AUTHOR]

The polymeric Ig receptor (pIgR) transports polymeric Abs across epithelia to the mucosa, where proteolytic cleavage releases the ectodomain (secretory component [SC]) as an integral component of secretory Abs, or as an unliganded protein that can mediate interactions with bacteria. SC is conserved among vertebrates, but domain organization is variable: mammalian SC has five domains (D1-D5), whereas avian, amphibian, and reptilian SC lack the D2 domain, and fish SC lacks domains D2-D4. In this study, we used double electron-electron resonance spectroscopy and surface plasmon resonance binding studies to characterize the structure, dynamics, and ligand binding properties of avian SC, avian SC domain variants, and a human SC (hSC) variant lacking the D2 domain. These experiments demonstrated that, unlike hSC, which adopts a compact or "closed" domain arrangement, unliganded avian SC is flexible and exists in both closed and open states, suggesting that the mammalian SC D2 domain stabilizes the closed conformation observed for hSC D1-D5. Experiments also demonstrated that avian and mammalian pIgR share related, but distinct, mechanisms of ligand binding. Together, our data reveal differences in the molecular recognition mechanisms associated with evolutionary changes in the pIgR protein.

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.

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.

Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

We used high hydrostatic pressure as a tool for exploring the conformational landscape of human cytochrome P450 3A4 (CYP3A4) by electron paramagnetic resonance and fluorescence spectroscopy. Site-directed incorporation of a luminescence resonance energy transfer donor-acceptor pair allowed us to identify a pressure-dependent equilibrium between two states of the enzyme, where an increase in pressure increased the spatial separation between the two distantly located fluorophores. This transition is characterized by volume change (ΔV°) and P1/2 values of -36.8 ± 5.0 mL/mol and 1.45 ± 0.33 kbar, respectively, which corresponds to a Keq° of 0.13 ± 0.06, so that only 15% of the enzyme adopts the pressure-promoted conformation at ambient pressure. This pressure-promoted displacement of the equilibrium is eliminated by the addition of testosterone, an allosteric activator. Using site-directed spin labeling, we demonstrated that the pressure- and testosterone-sensitive transition is also revealed by pressure-induced changes in the electron paramagnetic resonance spectra of a nitroxide side chain placed at position 85 or 409 of the enzyme. Furthermore, we observed a pressure-induced displacement of the emission maxima of a solvatochromic fluorophore (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin) placed at the same positions, which suggests a relocation to a more polar environment. Taken together, the results reveal an effector-dependent conformational equilibrium between open and closed states of CYP3A4 that involves a pronounced change at the interface between the region of α-helices A/A' and the meander loop of the enzyme, where residues 85 and 409 are located. Our study demonstrates the high potential of pressure-perturbation strategies for studying protein conformational landscapes.

As a first-line vertebrate immune defense, the polymeric immunoglobulin receptor (pIgR) transports polymeric IgA and IgM across epithelia to mucosal secretions, where the cleaved ectodomain (secretory component; SC) becomes a component of secretory antibodies, or when unliganded, binds and excludes bacteria. Here we report the 2.6Å crystal structure of unliganded human SC (hSC) and comparisons with a 1.7Å structure of teleost fish SC (tSC), an early pIgR ancestor. The hSC structure comprises five immunoglobulin-like domains (D1-D5) arranged as a triangle, with an interface between ligand-binding domains D1 and D5. Electron paramagnetic resonance measurements confirmed the D1-D5 interface in solution and revealed that it breaks upon ligand binding. Together with binding studies of mutant and chimeric SCs, which revealed domain contributions to secretory antibody formation, these results provide detailed models for SC structure, address pIgR evolution, and demonstrate that SC uses multiple conformations to protect mammals from pathogens.

G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.

Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron-electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼ 3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.

G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

Experimental techniques capable of determining the structure and dynamics of proteins are continuously being developed in order to understand protein function. Among existing methods, site-directed spin labeling in combination with saturation recovery (SR) electron paramagnetic resonance spectroscopy contributes uniquely to the determination of secondary and tertiary protein structure under physiological conditions, independent of molecular weight and complexity. In addition, SR of spin labeled proteins was recently demonstrated to be sensitive to conformational exchange events with characteristic lifetimes on the order of μs, a time domain that presents a significant challenge to other spectroscopic techniques. In this chapter, we present the theoretical background necessary to understand the capabilities of SR as applied to spin labeled proteins, the instrumental requirements, and practical experimental considerations necessary to obtain interpretable data, and the use of SR to obtain information on protein: (1) secondary structure via solvent accessibility measurements, (2) tertiary structure using interspin distance measurements, and (3) conformational exchange.

Site-directed spin labeling in combination with EPR is a powerful method for providing distances on the nm scale in biological systems. The most popular strategy, double electron-electron resonance (DEER), is carried out at cryogenic temperatures (50-80 K) to increase the short spin-spin relaxation time (T2) upon which the technique relies. A challenge is to measure long-range distances (20-60 Å) in proteins near physiological temperatures. Toward this goal we are investigating an alternative approach based on the distance-dependent enhancement of spin-lattice relaxation rate (T1(-1)) of a nitroxide spin label by a paramagnetic metal. With a commonly used nitroxide side chain (R1) and Cu(2+), it has been found that interspin distances ≤25 Å can be determined in this way (Jun et al. Biochemistry 2006, 45, 11666). Here, the upper limit of the accessible distance is extended to ≈40 Å using spin labels with long T1, a high-affinity 5-residue Cu(2+) binding loop inserted into the protein sequence, and pulsed saturation recovery to measure relaxation enhancement. Time-domain Cu(2+) electron paramagnetic resonance, quantum mechanical calculations, and molecular dynamics simulations provide information on the structure and geometry of the Cu(2+) loop and indicate that the metal ion is well-localized in the protein. An important aspect of these studies is that both Cu(2+)/nitroxide DEER at cryogenic temperatures and T1 relaxation measurements at room temperature can be carried out on the same sample, allowing both validation of the relaxation method and assessment of the effect of freezing on protein structure.

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell-cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities. Small changes in sequence are found to produce subtle structural and dynamical changes that impact relative affinities, in part via electrostatic and hydrophobic interactions, and in part through entropic effects because of increased conformational heterogeneity in the bound states as revealed by DEER distance mapping in the dimers. These findings highlight the remarkable ability of evolution to exploit a wide range of molecular properties to produce closely related members of the same protein family that have affinity differences finely tuned to mediate their biological roles.

Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins [bacterial microcompartment (in reference to the shell protein domain)] assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date.

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.

The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering.