Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy

Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native-like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60 ?-80 ?) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology.     

Determination of the orientation of T4 lysozyme vectorially bound to a planar-supported lipid bilayer using site-directed spin labeling

Site-directed spin labeling is used to investigate the structure of adsorbed T4 lysozyme (T4L). A monolayer of T4L is prepared by tethering the protein selectively via a His-tag to the chelating headgroups (NTA Ni) of a planar quartz-supported lipid bilayer. This results in a vectorially oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance spectra. Similar measurements of spin-labeled lipid bilayers were used to characterize the structure and dynamics of the supports. Electron paramagnetic resonance line shape was analyzed using the stochastic Liouville equation approach developed by Freed and co-workers. The simulations reveal a conservation of the secondary and tertiary structure of T4L upon adsorption although slight conformational changes in the presence of the surface can be detected by probing tertiary contact sites. The orientation of the entire protein was deduced on the basis of an anisotropic motional model for the spin-labeled side chain. In addition, a polar order but azimuthal disorder of the molecules was assumed to fit the data. These results demonstrate the utility of site-directed spin labeling in combination with spectral simulation to study not only the secondary and tertiary structure of adsorbed proteins in monolayer coverage but also their orientation with respect to the surface.     

pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efflux system

The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.     

Calculation of rigid-body conformational changes using restraint-driven Cartesian transformations

We present an approach for calculating conformational changes in membrane proteins using limited distance information. The method, named restraint-driven Cartesian transformations, involves 1) the use of relative distance changes; 2) the systematic sampling of rigid body movements in Cartesian space; 3) a penalty evaluation; and 4) model refinement using energy minimization. As a test case, we have analyzed the structural basis of activation gating in the Streptomyces lividans potassium channel (KcsA). A total of 10 pairs of distance restraints derived from site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) spectra were used to calculate the open conformation of the second transmembrane domains of KcsA (TM2). The SDSL-EPR based structure reveals a gating mechanism consistent with a scissoring-type motion of the TM2 segments that includes a pivot point near middle of the helix. The present approach considerably reduces the amount of time and effort required to establish the overall nature of conformational changes in membrane proteins. It is expected that this approach can be implemented into restrained molecular dynamics protocol to calculate the structure and conformational changes in a variety of membrane protein systems.     

High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane

We present a site-directed fluorescence labeling (SDFL) study of 25 different T4 lysozyme protein samples labeled with the thiol-cleavable fluorophore, (2-pyridyl)dithiobimane (PDT-Bimane). Our results demonstrate PDT-Bimane can be used in cysteine-scanning studies to detect protein secondary structure, and to map proximity between sites in proteins by monitoring tryptophan quenching of bimane fluorescence. In addition, the reducible nature of PDT-Bimane can be exploited to resolve problems often faced in SDFL studies: ensuring specific labeling of cysteine residues, determining the extent of free label contamination, and accurately determining labeling efficiency even at low concentrations. The ability to cleave PDT-Bimane off the protein enables rapid determination of these parameters, and positions it as an ideal fluorophore for automated, high-throughput structural studies of protein folding, the detection of protein-protein interactions, and the monitoring of real-time conformational changes.     

Coupling chemical biology and vibrational spectroscopy for studies of amyloids in vitro and in cells

Amyloid diseases are characterized by the aggregation of various proteins to form insoluble β-sheet–rich fibrils leading to cell death. Vibrational spectroscopies have emerged as attractive methods to study this process because of the rich structural information that can be extracted without large, perturbative probes. Importantly, specific vibrations such as the amide-I band directly report on secondary structure changes, which are key features of amyloid formation. Beyond intrinsic vibrations, the incorporation of unnatural vibrational probes can improve sensitivity for secondary structure determination (e.g. isotopic labeling), can provide residue-specific information of the surrounding polarity (e.g. unnatural amino acid), and are translatable into cellular studies. Here, we review the latest studies that have leveraged tools from chemical biology for the incorporation of novel vibrational probes into amyloidogenic proteins for both mechanistic and cellular studies.     

A new spin on protein dynamics

Site-directed spin labeling is a general method for investigating structure and conformational switching in soluble and membrane proteins. It will also be an important tool for exploring protein backbone dynamics. A semi-empirical analysis of nitroxide sidechain dynamics in spin-labeled proteins reveals contributions from fluctuations in backbone dihedral angles and rigid-body (collective) motions of alpha helices. Quantitative analysis of sidechain dynamics is sometimes possible, and contributions from backbone modes can be expressed in terms of relative order parameters and rates. Dynamic sequences identified by site-directed spin labeling correlate with functional domains, and so nitroxide scanning could provide an efficient strategy for identifying such domains in high-molecular weight proteins, supramolecular complexes and membrane proteins.     

Spin diffusion in globular proteins: alpha-lactalbumin

The structural properties of globular proteins analyzed by two different methods: high-resolution NMR and circular dichroism were compared. We established that the spin diffusion method shows changes in the secondary structure during the unfolding of the alpha-lactalbumin molten globule by urea. It was shown that the spin diffusion method is extremely effective in studies of interactions of water and denaturant molecules with the protein both in the native and the molten globule states.     

Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet

A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.     

Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors.

Changes in the amide bands in Fourier transform infrared spectra of proteins are generally attributed to alterations in protein secondary structure. In this study spectra of five different globular proteins were compared in the solid and solution states recorded with several sampling techniques. Spectral differences for each protein were observed between the various sampling techniques and physical states, which could not all be explained by a change in protein secondary structure. For example, lyophilization in the absence of lyoprotectants caused spectral changes that could (partially) have been caused by the removal of hydrating water molecules rather than secondary structural changes. Moreover, attenuated total reflectance spectra of proteins in H2O were not directly comparable to transmission spectra due to the anomalous dispersion effect. Our study also revealed that the amide I, II, and III bands differ in their sensitivities to changes in protein conformation: For example, strong bands in the region 1620-1630 and 1685-1695 cm(-1) were seen in the amide I region of aggregated protein spectra. Surprisingly, absorbance of such magnitudes was not observed in the amide II and III region. It appears, therefore, that only the amide I can be used to distinguish between intra- and intermolecular beta-sheet formation. Considering the differing sensitivity of the different amide modes to structural changes, it is advisable to utilize not only the amide I band, but also the amide II and III bands, to determine changes in protein secondary structure. Finally, it is important to realize that changes in these bands may not always correspond to secondary structural changes of the proteins.     

Domain selective labeling for NMR studies of multidomain proteins by domain ligation using highly active sortase A

Structural study of multidomain proteins using NMR is an emerging issue for understanding biological functions. To this end, domain-specific labeling is expected to be a key technology for facilitating the NMR-assignment process and for collecting distance information via spin labeling. To obtain domain-specific labeled samples, use of sortase A as a protein ligation tool is a viable approach. Sortase A enables ligation of separately expressed proteins (domains) through the Leu-Pro-X-Thr-Gly linker. However, the ligation reaction mediated by sortase A is not efficient. Poor yield and long reaction times hamper large-scale preparation using sortase A. Here we report the application of highly active sortases to NMR analyses. Optimal yields can be achieved within several hours when the ligation reaction are mediated by highly active sortases at 4 °C. We propose that this protocol can contribute to structural analyses of multidomain proteins by NMR.     

Fourier transform infrared spectroscopic analysis of protein secondary structures

Infrared spectroscopy is one of the oldest and well established experimental techniques for the analysis of secondary structure of polypeptides and proteins. It is convenient, non-destructive, requires less sample preparation, and can be used under a wide variety of conditions. This review introduces the recent developments in Fourier transform infrared （FTIR） spectroscopy technique and its applications to protein structural studies. The experimental skills, data analysis, and correlations between the FTIR spectroscopic bands and protein secondary structure components are discussed. The applications of FTIR to the second- ary structure analysis, conformational changes, structural dynamics and stability studies of proteins are also discussed.     

Global structural changes in annexin 12. The roles of phospholipid,Ca2+, and pH

Ca2+-dependent membrane interaction has long been recognized as a general property of the annexin (ANX) family of proteins. More recently, it has become clear that ANXs can also undergo Ca2+-independent membrane interactions at mildly acidic pH. Here we use site-directed spin labeling in combination with circular dichroism and biochemical labeling methods to compare the structure and membrane topography of these two different membrane-bound forms of ANX12. Our results reveal strong similarities between the solution structure and the structure of the Ca2+-dependent membrane-bound form at neutral pH. In contrast, all Ca2+-independent membrane interactions tested resulted in large scale conformational changes and membrane insertion. Pairs of spin labels that were in close proximity across the interface of different domains of the protein in both the soluble and Ca2+-dependent membrane form were >25 A apart in the Ca2+-independent membrane-bound form. Despite these major conformational changes, the overall secondary structure content did not appear to be strongly altered and ANX12 remained largely helical. Thus, Ca2+-independent membrane interaction leads to massive refolding but not unfolding. Refolding did not occur at low pH in the absence of membranes but occurred within a few seconds after phospholipid vesicles were added. The phospholipid composition of the vesicles was an important modulator of Ca2+-independent membrane interaction. For example, cardiolipin-containing vesicles induced Ca2+-independent membrane interaction even at near neutral pH, thereby raising the possibility that lipid composition could induce relatively rapid Ca2+-independent membrane interaction in vivo.     

EPR spin labeling measurements of thylakoid membrane fluidity during barley leaf senescence

Physical properties of thylakoid membranes isolated from barley were investigated by the electron paramagnetic resonance (EPR) spin labeling technique. EPR spectra of stearic acid spin labels 5-SASL and 16-SASL were measured as a function of temperature in secondary barley leaves during natural and dark-induced senescence. Oxygen transport parameter was determined from the power saturation curves of the spin labels obtained in the presence and absence of molecular oxygen at 25 °C. Parameters of EPR spectra of both spin labels showed an increase in the thylakoid membrane fluidity during senescence, in the headgroup area of the membrane, as well as in its interior. The oxygen transport parameter also increased with age of barley, indicating easier diffusion of oxygen within the membrane and its higher fluidity. The data are consistent with age-related changes of the spin label parameters obtained directly by EPR spectroscopy. Similar outcome was also observed when senescence was induced in mature secondary barley leaves by dark incubation. Such leaves showed higher membrane fluidity in comparison with leaves of the same age, grown under light conditions. Changes in the membrane fluidity of barley secondary leaves were compared with changes in the levels of carotenoids (car) and proteins, which are known to modify membrane fluidity. Determination of total car and proteins showed linear decrease in their level with senescence. The results indicate that thylakoid membrane fluidity of barley leaves increases with senescence; the changes are accompanied with a decrease in the content of car and proteins, which could be a contributing factor.     

Tertiary structure variability within the quaternary states of hemoglobin: a spin label study

Using variable temperature techniques, the spin label spectral resolution of hemoglobin labeled at the beta93 cysteines with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodonacetamide has been greatly enhanced. The effects of different ligands, inositol hexaphosphate, pH and salt concentration upon spin labeled ferrous and ferric hemoglobin indicate that the beta chain tertiary structure exhibits considerable variability within the oxy and deoxy quaternary structures. From these studies ligand and spin state changes both appear to be of significance in producing structural changes; binding of inositol hexaphosphate then produces further structural changes secondary in amplitude.     

Isotope-labeled vibrational circular dichroism studies of calmodulin and its interactions with ligands

In this work we have studied ligand-induced secondary structure changes in the small calcium regulatory protein calmodulin (CaM) using vibrational circular dichroism (VCD) spectroscopy. We find that, due to its chiral sensitivity, VCD spectroscopy has increased ability over IR spectroscopy to detect changes in the structure and flexibility of secondary structure elements upon ligand binding. Moreover, we demonstrate that the uniform isotope labeling of CaM with (13)C shifts its amide I' VCD band by about approximately 43 cm(-1) to lower wavenumbers, which opens up a spectral window to simultaneously visualize a bound target protein. Therefore this study also provides the first example of how isotope labeling enables protein-protein interactions to be studied by VCD with good separation of the signals for both isotope-labeled and unlabeled proteins.     

Algorithm for selection of optimized EPR distance restraints for de novo protein structure determination

A hybrid protein structure determination approach combining sparse Electron Paramagnetic Resonance (EPR) distance restraints and Rosetta de novo protein folding has been previously demonstrated to yield high quality models (Alexander et al. (2008)). However, widespread application of this methodology to proteins of unknown structures is hindered by the lack of a general strategy to place spin label pairs in the primary sequence. In this work, we report the development of an algorithm that optimally selects spin labeling positions for the purpose of distance measurements by EPR. For the α-helical subdomain of T4 lysozyme (T4L), simulated restraints that maximize sequence separation between the two spin labels while simultaneously ensuring pairwise connectivity of secondary structure elements yielded vastly improved models by Rosetta folding. 54% of all these models have the correct fold compared to only 21% and 8% correctly folded models when randomly placed restraints or no restraints are used, respectively. Moreover, the improvements in model quality require a limited number of optimized restraints, which is determined by the pairwise connectivities of T4L α-helices. The predicted improvement in Rosetta model quality was verified by experimental determination of distances between spin labels pairs selected by the algorithm. Overall, our results reinforce the rationale for the combined use of sparse EPR distance restraints and de novo folding. By alleviating the experimental bottleneck associated with restraint selection, this algorithm sets the stage for extending computational structure determination to larger, traditionally elusive protein topologies of critical structural and biochemical importance.     

De novo high-resolution protein structure determination from sparse spin-labeling EPR data

As many key proteins evade crystallization and remain too large for nuclear magnetic resonance spectroscopy, electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling offers an alternative approach for obtaining structural information. Such information must be translated into geometric restraints to be used in computer simulations. Here, distances between spin labels are converted into distance ranges between beta carbons by using a "motion-on-a-cone" model, and a linear-correlation model links spin-label accessibility to the number of neighboring residues. This approach was tested on T4-lysozyme and alphaA-crystallin with the de novo structure prediction algorithm Rosetta. The results demonstrate the feasibility of obtaining highly accurate, atomic-detail models from EPR data by yielding 1.0 A and 2.6 A full-atom models, respectively. Distance restraints between amino acids far apart in sequence but close in space are most valuable for structure determination. The approach can be extended to other experimental techniques such as fluorescence spectroscopy, substituted cysteine accessibility method, or mutational studies.     

Secondary structure-based assignment of the protein structural classes

Structural class categorizes proteins based on the amount and arrangement of the constituent secondary structures. The knowledge of structural classes is applied in numerous important predictive tasks that address structural and functional features of proteins. We propose novel structural class assignment methods that use one-dimensional (1D) secondary structure as the input. The methods are designed based on a large set of low-identity sequences for which secondary structure is predicted from their sequence (PSSA^sc model) or assigned based on their tertiary structure (SSA^sc). The secondary structure is encoded using a comprehensive set of features describing count, content, and size of secondary structure segments, which are fed into a small decision tree that uses ten features to perform the assignment. The proposed models were compared against seven secondary structure-based and ten sequence-based structural class predictors. Using the 1D secondary structure, SSA^sc and PSSA^sc can assign proteins to the four main structural classes, while the existing secondary structure-based assignment methods can predict only three classes. Empirical evaluation shows that the proposed models are quite promising. Using the structure-based assignment performed in SCOP (structural classification of proteins) as the golden standard, the accuracy of SSA^sc and PSSA^sc equals 76 and 75%, respectively. We show that the use of the secondary structure predicted from the sequence as an input does not have a detrimental effect on the quality of structural class assignment when compared with using secondary structure derived from tertiary structure. Therefore, PSSA^sc can be used to perform the automated assignment of structural classes based on the sequences.