Site-directed Spin Labeling Technique Research Articles

Effect Of Type I Antifreeze Proteins On The Freezing And Melting Processes Of Cryoprotective Solutions Studied By Site Directed Spin Labeling Technique

Effect Of Type I Antifreeze Proteins On The Freezing And Melting Processes Of Cryoprotective Solutions Studied By Site Directed Spin Labeling Technique

EPR Techniques to Probe Insertion and Conformation of Spin-Labeled Proteins in Lipid Bilayers.

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, for example, in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance (NMR) spectroscopy, having only one or few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with an exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity and data interpretation, thus it is difficult to give ready-to-go methodological recipes. However, new technological developments (arbitrary waveform generators) and new spin labels spectroscopically orthogonal to nitroxides increased the range of applicability from in vitro toward in-cell EPR experiments. This chapter is an updated version of the one published in the first edition of the book and describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, addressing new tools such as arbitrary waveform generators and spectroscopically orthogonal labels, such as Gd(III)-based labels. We will present challenges in sample preparation and data analysis for functional and structural membrane protein studies using site-directed spin labeling techniques and give experimental details on EPR techniques providing information on side chain dynamics and water accessibility using nitroxide probes. An updated optimal Q-band DEER setup for nitroxide probes will be described, and its extension to gadolinium-containing samples will be addressed.

Effect of Type I Antifreeze Proteins on the Freezing and Melting Processes of Cryoprotective Solutions Studied by Site-Directed Spin Labeling Technique.

Antifreeze proteins (AFPs) protect organisms living in subzero environments from freezing injury, which render them potential applications for cryopreservation of living cells, organs, and tissues. Cryoprotective agents (CPAs), such as glycerol and propylene glycol, have been used as ingredients to treat cellular tissues and organs to prevent ice crystal’s formation at low temperatures. To assess AFP’s function in CPA solutions, we have the applied site-directed spin labeling technique to a Type I AFP. A two-step process to prevent bulk freezing of the CPA solutions was observed by the cryo-photo microscopy, i.e., (1) thermodynamic freezing point depression by the CPAs; and (2) inhibition to the growth of seed ice crystals by the AFP. Electron paramagnetic resonance (EPR) experiments were also carried out from room temperature to 97 K, and vice versa. The EPR results indicate that the spin labeled AFP bound to ice surfaces, and inhibit the growths of ice through the bulk freezing processes in the CPA solutions. The ice-surface bound AFP in the frozen matrices could also prevent the formation of large ice crystals during the melting processes of the solutions. Our study illustrates that AFPs can play an active role in CPA solutions for cryopreservation applications.

New insight of antifreeze proteins via site-directed spin labeling technique

New insight of antifreeze proteins via site-directed spin labeling technique

Mechanisms of antifreeze proteins investigated via the site-directed spin labeling technique.

The site-directed spin labeling (SDSL) technique was used to examine the antifreeze mechanisms of type-I antifreeze proteins (AFPs). The effects on the growth of seed ice crystals by the spin-label groups attached to different side chains of the AFPs were observed, and the states of water molecules surrounding the spin-label groups were probed via analyses of variable-temperature (VT) dependent electron paramagnetic resonance (EPR) spectra. The first set of experiments revealed the antifreeze activities of the spin-labeled AFPs at the microscopic level, while the second set of experiments displayed those at the molecular level. The experimental results confirmed the putative ice-binding surface (IBS) of type-I AFPs. The VT EPR spectra indicate that type-I AFPs can inhibit the nucleation of seed ice crystals down to~-20°C in their aqueous solutions. Thus, the present authors believe that AFPs protect organisms from freezing damage in two ways: (1) inhibiting the nucleation of seed ice crystals, and (2) hindering the growth of seed ice crystals once they have formed. The first mechanism should play a more significant role in protecting against freezing damage among organisms living in cold environments. The VT EPR spectra also revealed that liquid-like water molecules existed around the spin-labeled non-ice-binding side chains of the AFPs frozen within the ice matrices, and ice surrounding the spin-label groups melted at subzero temperatures during the heating process. This manuscript concludes with the proposed model of antifreeze mechanisms of AFPs based on the experimental results.

Antifreeze Mechanism Study of Ice Growth Inhibition by Spin Labeled Ice Binding Proteins

Ice binding proteins (IBPs) were found to play critical role for various organisms to survive in extreme cold climates. We carried out the study on spin labeled type 1 IBPs whose sequence is from that of the Pseudopleuronectes americanus (winter flounder). The spin labeled sites involved in our study have undergone a manipulation on different directions along the α-helical structure. Site directed spin labeling technique was used to make the spin labeled peptides. Combination of the EPR spectra at low temperatures and observation of their inhibition to the growth of ice crystals gives us insight into their antifreeze mechanism on both molecular scale and microscopic scale. This poster will focus on presenting the experimental result of ice crystal inhibition, while another poster of my fellow student researchers will focus on the EPR result. Osmometer-microscopy technique was used to observe the ice crystals. The changes in antifreeze activities, kinetics of ice crystals’ growths and shapes of the ice crystals by these spin labeled IBPs compared with the wild type IBP were observed and will be discussed in this poster. Results include observations of IBP binding on both sides of the basal plane and prism faces of ice seed crystals. Significant differences between thermal hysteresis and crystal structured due to the binding of the Spin labeled IBPs were observed which is correlated to the EPR spectra at molecular level. Single cystal growth on the horizontal axis is observed within the hysteresis gap of the spin labeled IBPs while the wild type IBP showed no crystal growth during the hysteresis gap. There were various differences between thermal hysteresis values between the different spin labeled IBPs.

Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label.

Guanine-rich oligonucleotides can form a unique G-quadruplex (GQ) structure with stacking units of four guanine bases organized in a plane through Hoogsteen bonding. GQ structures have been detected in vivo and shown to exert their roles in maintaining genome integrity and regulating gene expression. Understanding GQ conformation is important for understanding its inherent biological role and for devising strategies to control and manipulate functions based on targeting GQ. Although a number of biophysical methods have been used to investigate structure and dynamics of GQs, our understanding is far from complete. As such, this work explores the use of the site-directed spin labeling technique, complemented by molecular dynamics simulations, for investigating GQ conformations. A nucleotide-independent nitroxide label (R5), which has been previously applied for probing conformations of noncoding RNA and DNA duplexes, is attached to multiple sites in a 22-nucleotide DNA strand derived from the human telomeric sequence (hTel-22) that is known to form GQ. The R5 labels are shown to minimally impact GQ folding, and inter-R5 distances measured using double electron-electron resonance spectroscopy are shown to adequately distinguish the different topological conformations of hTel-22 and report variations in their occupancies in response to changes of the environment variables such as salt, crowding agent, and small molecule ligand. The work demonstrates that the R5 label is able to probe GQ conformation and establishes the base for using R5 to study more complex sequences, such as those that may potentially form multimeric GQs in long telomeric repeats.

Analyzing the Viability of Various Native Membrane Mimics for Membrane Proteins using Site-Directed Spin Labeling EPR

Membrane proteins have become the target for the majority of drug related therapies in recent years; in contrast, there is limited information available on membrane proteins due to the difficulties of studying them in vitro. In order to study their structure and function, it is crucial to prepare suitable, native-like membrane mimics. Our studies involve KCNE1, a transmembrane protein located in the heart that modulates the activity of the KCNQ1 voltage-gated potassium channel. An important protein for proper cardiac function, mutations in the structure can lead to atrial fibrillation, long QT syndrome, and deafness. In order to assess the viability of various membrane mimics for studying membrane proteins, we have utilized site-directed spin labeling (SDSL)and electron paramagnetic resonance (EPR) spectroscopic techniques, which are well established methods of studying protein structure. The CW-EPR spectral line shape analysis was conducted on an inside probe (F56C) and outside probe (R33C) in various vesicle compositions (POPC, POPG, DMPC, DOPC, DPPC, and DOPG) in order to assess the accuracy and precision of various membrane mimics. This study will provide a path for researchers working on membrane protein EPR spectroscopic studies to select a better membrane mimetic environment.

High-field ELDOR-detected NMR study of a nitroxide radical in disordered solids: Towards characterization of heterogeneity of microenvironments in spin-labeled systems

The combination of high-field EPR with site-directed spin-labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in probing the polarity and proticity characteristics of protein/matrix systems. This information is concluded from the principal components of the nitroxide Zeeman (g), nitrogen hyperfine (A) and quadrupole (P) tensors of the spin labels attached to specific sites. Recent multi-frequency high-field EPR studies underlined the complexity of the problem to treat the nitroxide microenvironment in proteins adequately due to inherent heterogeneities which result in several principal x-components of the nitroxide g-tensor. Concomitant, but distinctly different nitrogen hyperfine components could, however, not be determined from high-field cw EPR experiments owing to the large intrinsic EPR linewidth in fully protonated guest/host systems. It is shown in this work that, using the W-band (95GHz) ELDOR- (electron–electron double resonance) detected NMR (EDNMR) method, different principal nitrogen hyperfine, Azz, and quadrupole, Pzz, tensor values of a nitroxide radical in glassy 2-propanol matrix can be measured with high accuracy. They belong to nitroxides with different hydrogen-bond situations. The satisfactory resolution and superior sensitivity of EDNMR as compared to the standard ENDOR (electron–nuclear double resonance) method are demonstrated.

Incorporation of a Rigid TOAC Spin-Label as a Non-Native Amino Acid into a Full-Length Protein by In Vitro Translation using Amber Codon Suppression

The development of EPR site-directed spin-labeling (SDSL) techniques has provided a powerful avenue for the study of membrane protein structure and dynamics. One of the advantages of EPR is the ability to obtain distance and dynamic information about particular protein regions. The accuracy of this information is highly dependent on the spin-label that is used. Labels with less inherent mobility are better reporters of protein backbone dynamics and give more sharply defined distance parameters. TOAC is a commonly used rigid spin-label that can be integrated into the protein backbone as an amino acid analog. However, its current use is limited to the study of small peptides that can be only generated synthetically (solid-phase peptide synthesis). A new biochemical approach for incorporation of non-natural amino acids during translation using a synthetically loaded amber suppressor tRNA has successfully generated TOAC-labeled protein but not in sufficient yields for EPR studies. Here, we describe the use of an amber codon suppressor tRNA and a continuous exchange cell-free (CECF) expression system for in vitro translation of a small thylakoid membrane protein, Tha4, containing site-specific integration of TOAC. Further, we demonstrate successful incorporation of the TOAC-containing Tha4 into liposomes by supplying POPC vesicles during the translation reaction. By combining new techniques in suppressor tRNA and continuous exchange cell-free (CECF) translation, we have developed a method for translation of full length proteins with site-directed insertion of non-natural amino acid spin labels in yields applicable to CW-EPR experiments.

Conformational Distributions at the N-peptide/boxB RNA Interface Studied Using Site-Directed Spin Labeling

In bacteriophage lambda, interactions between a 22-amino acid peptide (called the N-peptide) and a stem-loop RNA element (called boxB) play a critical role in transcription anti-termination. The N-peptide/boxB complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/RNA recognition. Particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the boxB RNA. The studies have led to a model in which the bound N-peptide exists in dynamic equilibrium between two states, with peptide C-terminal fragment either stacking on (i.e., the stacked state) or peeling away from (i.e., the unstacked state) the RNA loop. The function of the N-peptide/boxB complex seems to correlate with the fraction of the stacked state. Here, the N-peptide/boxB system is studied using the site-directed spin labeling technique, in which X-band electron paramagnetic resonance spectroscopy is applied to monitor nanosecond rotational behaviors of stable nitroxide radicals covalently attached to different positions of the N-peptide. The data reveal that in the nanosecond regime the C-terminal fragment of bound N-peptide adopts multiple discrete conformations within the complex. The characteristics of these conformations are consistent with the proposed stacked and unstacked states, and their distributions vary upon mutations within the N-peptide. These results suggest that the dynamic two-state model remains valid in the nanosecond regime, and represents a unique mode of function in the N-peptide/boxB RNA complex. It also demonstrates a connection between picosecond and nanosecond dynamics in a biological complex.

Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling

In bacteriophage λ, interactions between a 22-amino acid peptide (called the N-peptide) and a stem-loop RNA element (called boxB) play a critical role in transcription anti-termination. The N-peptide/boxB complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/RNA recognition. Particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the boxB RNA. The studies have led to a model in which the bound N-peptide exists in dynamic equilibrium between two states, with peptide C-terminal fragment either stacking on (i.e., the stacked state) or peeling away from (i.e., the unstacked state) the RNA loop. The function of the N-peptide/boxB complex seems to correlate with the fraction of the stacked state. Here, the N-peptide/boxB system is studied using the site-directed spin labeling technique, in which X-band electron paramagnetic resonance spectroscopy is applied to monitor nanosecond rotational behaviors of stable nitroxide radicals covalently attached to different positions of the N-peptide. The data reveal that in the nanosecond regime the C-terminal fragment of bound N-peptide adopts multiple discrete conformations within the complex. The characteristics of these conformations are consistent with the proposed stacked and unstacked states, and their distributions vary upon mutations within the N-peptide. These results suggest that the dynamic two-state model remains valid in the nanosecond regime, and represents a unique mode of function in the N-peptide/boxB RNA complex. It also demonstrates a connection between picosecond and nanosecond dynamics in a biological complex.

A Nucleotide-Independent Nitroxide Probe Reports on Site-Specific Stereomeric Environment in DNA

In this report, stereospecific structural and dynamic features in DNA are studied using the site-directed spin labeling technique. A stable nitroxide radical, 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), was attached postsynthetically to phosphorothioates that were chemically introduced, one at a time, at five sites of a DNA duplex. The two phosphorothioate diastereomers (R p or S p ) were separated, and nitroxide rotational motions were monitored using electron paramagnetic resonance spectroscopy. The resulting spectra vary according to diastereomer identity and location of the labeling site, with R p -R5a spectra effectively reporting on local DNA structural features and S p -R5a spectra sensing variations in local DNA motions. This establishes R p - and S p -R5a as unique probes for investigating nucleic acids in a site- and stereospecific manner, which may aid studies of stereospecific DNA/protein interactions. In addition, weighted averages of individual R p and S p spectra match those of R5a attached to mixed diastereomers. This suggests that R5a linked to mixed diastereomers reports on the composite behaviors of R p - and S p -R5a and is useful in initial probing of the DNA local environment. This work advances understanding of R5a/DNA coupling, and is a key step forward in developing a nucleotide-independent spectroscopic probe for studying nucleic acids.

Dynamics and Local Ordering of Spin-Labeled Prion Protein: An ESR Simulation Study of a Highly PH-Sensitive Site

Valine 160 on β-sheet-2 (S2) of mouse prion (moPrPC) has been previously identified as the most highly pH-sensitive site on moPrPC by ESR spectroscopy using site-directed spin labeling (SDSL) technique. However, no further theoretical analysis to reveal the molecular dynamics reported on the experimental ESR spectra is available. The X-band ESR spectra of R1 nitroxide spin label at V160 and four other sites are carefully analyzed over large pH and temperature ranges using a spectral simulation method based upon stochastic Liouville equation (SLE). The results clearly reveal the dynamics and ordering of the local environment of V160R1 showing that (i) molecular mobility of V160R1 on S2 gradually increases with a decrease of pH from 7.5 to 4.5; (ii) two distinctly different spectral components are simultaneously present in all spectra of V160R1 studied. The spectral components are, respectively, denoted as immobile (Im), characterized by lower molecular mobility and higher ordering, and mobile (Mb) component of high mobility and low ordering. The population ratio (Im/Mb) increases with increasing pH, while Im remains dominant in all V160R1 spectra. It suggests a more mobile and disordered dynamic molecular structure for mouse PrPC, which is very likely correlated with increased β-sheet content at low pH, as the environment changes from neutral to acidic pH. Together with the results of the SLE-based analyses on the spectra of other sites that appear pH-insensitive, we suggest that the simultaneous presence of the spectral components for V160R1 is strongly correlated with the coexistence of multiple protein conformations in local structure of PrPC over the varied pH range. It demonstrates that the combined approach of the SDSL technique and the SLE-based analysis leads to a powerful method for unraveling the complexity of protein dynamics.

High-Field EPR and ESEEM Investigation of the Nitrogen Quadrupole Interaction of Nitroxide Spin Labels in Disordered Solids: Toward Differentiation between Polarity and Proticity Matrix Effects on Protein Function

The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.

Molecular Dynamics Simulation of Site-Directed Spin Labeling: Experimental Validation in Muscle Fibers

We have developed a computational molecular dynamics technique to simulate the motions of spin labels bound to the regulatory domain of scallop myosin. These calculations were then directly compared with site-directed spin labeling experimental results obtained by preparing seven single-cysteine mutants of the smooth muscle (chicken gizzard) myosin regulatory light chain and performing electron paramagnetic resonance experiments on these spin-labeled regulatory light chains in functional scallop muscle fibers. We determined molecular dynamics simulation conditions necessary for obtaining a convergent orientational trajectory of the spin label, and from these trajectories we then calculated correlation times, orientational distributions, and order parameters. Simulated order parameters closely match those determined experimentally, validating our molecular dynamics modeling technique, and demonstrating our ability to predict preferred sites for labeling by computer simulation. In several cases, more than one rotational mode was observed within the 14-ns trajectory, suggesting that the spin label samples several local energy minima. This study uses molecular dynamics simulations of an experimental system to explore and enhance the site-directed spin labeling technique.

Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.

Guanidine hydrochloride unfolding of a transmembrane beta-strand in FepA using site-directed spin labeling.

We have used the electron spin resonance (ESR) site-directed spin-labeling (SDSL) technique to examine the guanidine hydrochloride (Gdn-HCl) induced denaturation of several sites along a transmembrane beta-strand located in the ferric enterobactin receptor, FepA. In addition, we have continued the characterization of the beta-strand previously identified by our group (Klug CS et al., 1997, Biochemistry 36:13027-13033) to extend from the periplasm to the extracellular surface loop in FepA, an integral membrane protein containing a beta-barrel motif comprised of a series of antiparallel beta-strands that is responsible for transport of the iron chelate, ferric enterobactin (FeEnt), across the outer membrane of Escherichia coli and many related enteric bacteria. We have previously shown that a large surface loop in FepA containing the FeEnt binding site denatures independently of the beta-barrel domain (Klug CS et al., 1995, Biochemistry 34:14230-14236). The SDSL approach allows examination of the unfolding at individual residues independent of the global unfolding of the protein. This work shows that sites along the beta-strand that are exposed to the aqueous lumen of the channel denature more rapidly and with higher cooperativity than the surface loop, while sites on the hydrophobic side of the beta-strand undergo a limited degree of noncooperative unfolding and do not fully denature even at high (e.g., 4 M) Gdn-HCl concentrations. We conclude that, in a transmembrane beta-strand, the local environment of a given residue plays a significant role in the loss of structure at each site.