RESUMEN
Proteins tethered to solid supports are of increasing interest in bioanalytical chemistry and protein science in general. However, the extent to which tethering modifies the energy landscape and dynamics of the protein is most often unknown because there are few biophysical methods that can determine secondary and tertiary structures and explore conformational equilibria and dynamics of a tethered protein with site-specific resolution. Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) offers a unique opportunity for this purpose. Here, we employ a general strategy using unnatural amino acids that enables efficient and site-specific tethering of a spin-labeled protein to a Sepharose solid support. Remarkably, EPR spectra of spin-labeled T4 lysozyme (T4L) reveal that a single site-specific attachment suppresses rotational motion of the protein sufficiently to allow interpretation of the spectral line shape in terms of protein internal dynamics. Importantly, line shape analysis and distance mapping using double electron-electron resonance reveal that internal dynamics, the tertiary fold, conformational equilibria, and ligand binding of the tethered proteins were similar to those in solution, in contrast to random attachment via native lysine residues. The results of this study set the stage for the development of an EPR-based flow system that will house soluble and membrane proteins immobilized site-specifically, thereby enabling facile screening of structural and dynamical effects of binding partners.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Muramidasa/química , Marcadores de Spin , Sitios de Unión/fisiología , Muramidasa/metabolismo , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
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 ß-strand, or with cysteine residues in adjacent strands in a ß-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.
Asunto(s)
Óxidos de Nitrógeno/química , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Conformación ProteicaRESUMEN
The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each containing a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amounts qualitatively similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants containing the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in solution. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.
Asunto(s)
Fenilalanina/análogos & derivados , Marcadores de Spin , Modelos Moleculares , Mutación , Fenilalanina/química , Fenilalanina/genéticaRESUMEN
The measurement of (1)H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed α-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed α-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.
Asunto(s)
Calmodulina/química , Disulfuros/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Muramidasa/química , Bacteriófago T4/enzimología , Óxidos N-Cíclicos/síntesis química , Óxidos N-Cíclicos/química , Humanos , Modelos Moleculares , Conformación Proteica , Marcadores de Spin/síntesis químicaRESUMEN
We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.
Asunto(s)
Muramidasa/metabolismo , Proteínas Virales/metabolismo , Bacteriófago T4/enzimología , Bacteriófago T4/genética , Biocatálisis , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Simulación de Dinámica Molecular , Método de Montecarlo , Muramidasa/química , Muramidasa/genética , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Long interspersed nuclear element-1 is a highly abundant mammalian retrotransposon that comprises 17% of the human genome. L1 retrotransposition requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. ORF1p has been shown to adopt a homo-trimeric, asymmetric dumbbell-shaped structure. However, its atomic-level structure and mechanism of RNA binding remains poorly understood. Here, we report the results of a site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) study of 27 residues within the RNA binding region of the full-length protein. The EPR data are compatible with the large RNA binding lobe of ORF1p containing a RNA recognition motif (RRM) domain and a carboxyl-terminal domain (CTD) that are predicted from crystallographic and NMR studies of smaller fragments of the protein. Interestingly, the EPR data indicate that residues in strands ß3 and ß4 of the RRM are structurally unstable, compatible with the previously observed sensitivity of this region to proteolysis. Affinity measurements and RNA-dependent EPR spectral changes map the RNA binding site on ORF1p to residues located in strands ß3 and ß4 of the RRM domain and to helix α1 of the CTD. Complementary in vivo studies also identify residues within the RRM domain that are required for retrotransposition. We propose that in the context of the full-length trimeric protein these distinct surfaces are positioned adjacent to one another providing a continuous surface that may interact with nucleic acids.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Elementos de Nucleótido Esparcido Largo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencia de Aminoácidos , Animales , Endonucleasas/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , ADN Polimerasa Dirigida por ARN/metabolismo , Alineación de SecuenciaRESUMEN
An extensive set of electron spin resonance spectra was obtained over a wide range of frequencies (9, 95, 170, and 240 GHz) and temperatures (2 to 32 degrees C) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model, the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2 are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered.
Asunto(s)
Bacteriófago T4/enzimología , Muramidasa/química , Marcadores de Spin , Espectroscopía de Resonancia por Spin del Electrón , Peso Molecular , Movimiento (Física) , Muramidasa/genética , Muramidasa/metabolismo , Mutagénesis Sitio-Dirigida , Óxidos de Nitrógeno/química , Óxidos de Nitrógeno/metabolismo , Conformación Proteica , Sacarosa/química , Temperatura , Agua/químicaRESUMEN
A disulfide-linked nitroxide side chain (R1) used in site-directed spin labeling of proteins often exhibits an EPR spectrum characteristic of a weakly ordered z-axis anisotropic motion at topographically diverse surface sites, including those on helices, loops and edge strands of beta-sheets. To elucidate the origin of this motion, the first crystal structures of R1 that display simple z-axis anisotropic motion at solvent-exposed helical sites (131 and 151) and a loop site (82) in T4 lysozyme have been determined. Structures of 131R1 and 151R1 determined at cryogenic or ambient temperature reveal an intraresidue C(alpha)--H...S(delta) interaction that immobilizes the disulfide group, consistent with a model in which the internal motions of R1 are dominated by rotations about the two terminal bonds (Columbus, Kálai, Jeko, Hideg, and Hubbell, Biochemistry 2001;40:3828-3846). Remarkably, the 131R1 side chain populates two rotamers equally, but the EPR spectrum reflects a single dominant dynamic population, showing that the two rotamers have similar internal motion determined by the common disulfide-backbone interaction. The anisotropic motion for loop residue 82R1 is also accounted for by a common disulfide-backbone interaction, showing that the interaction does not require a specific secondary structure. If the above observations prove to be general, then significant variations in order and rate for R1 at noninteracting solvent-exposed helical and loop sites can be assigned to backbone motion because the internal motion is essentially constant.
Asunto(s)
Óxidos de Nitrógeno/química , Proteínas/química , Marcadores de Spin , Bacteriófago T4/enzimología , Cristalografía por Rayos X , Disulfuros/química , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Muramidasa/química , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Temperatura , Proteínas Virales/químicaRESUMEN
Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin-labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site-directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native-like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/química , Muramidasa/química , Mioglobina/química , Rodopsina/química , Animales , Ficoll/química , Conformación Proteica , Ratas , Cachalote , Marcadores de Spin , Sacarosa/químicaRESUMEN
Previous studies of symmetry preferences in protein crystals suggest that symmetric proteins, such as homodimers, might crystallize more readily on average than asymmetric, monomeric proteins. Proteins that are naturally monomeric can be made homodimeric artificially by forming disulfide bonds between individual cysteine residues introduced by mutagenesis. Furthermore, by creating a variety of single-cysteine mutants, a series of distinct synthetic dimers can be generated for a given protein of interest, with each expected to gain advantage from its added symmetry and to exhibit a crystallization behavior distinct from the other constructs. This strategy was tested on phage T4 lysozyme, a protein whose crystallization as a monomer has been studied exhaustively. Experiments on three single-cysteine mutants, each prepared in dimeric form, yielded numerous novel crystal forms that cannot be realized by monomeric lysozyme. Six new crystal forms have been characterized. The results suggest that synthetic symmetrization may be a useful approach for enlarging the search space for crystallizing proteins.