Electron paramagnetic resonance study of the interactions between steroid hormones and binding proteins]

Interaction of a spin labeled corticosteroid (desoxycorticosterone nitroxyde: DOC -NO) with three purified proteins (albumin, transcortin, progesterone binding protein: PBG) was studied by electron spin resonance (ESR) spectroscopy. DOC-NO was competitive with natural corticosteroids and therefore bound at the same site to specific binding proteins. ESR spectra in the presence of each of the proteins showed an immobilized (bound) form of the spin labeled steroid and allowed the calculation of the corresponding association constant (Ka) at equilibrium. The three binding proteins could be characterized by the ESR parameters of the DOC-NO bound form. The thermodynamic parameters (deltaH, deltaS) of the steroid-protein interactions were calculated from the ESR data obtained within a wide temperature range (3--40 degrees C). The ESR spectra width (2T) was used to evaluate the polarity of the spin label environment within the steroid binding site: a hydrophobic character was observed for transcortin whereas PBG exhibited a more hydrophilic steroid binding sits. The rotational correlation time of the three protein DOC-NO complexes at equilibrium were calculated from ESR data; the results were correlated with the protein molecular size and suggested a non spherical shape for the binding macromolecule in solution. Spin labelling of biologically active steroids thus provides a novel approach for the study of the interaction of these hormones with their binding protein. Providing a suitable spin label, the ESR parameters may allow the characterization of several types of binding sites of different biological significance for the same hormone, in biological fluids as well as in target tissues.     

Electron spin resonance study of human alpha 1-acid glycoprotein interaction with a spin labelled steroid.

The interaction of human alpha 1-acid glycoprotein (AAG) with a corticosteroid was studied using nitroxide labeled deoxycorticosterone and electron spin resonance (ESR) spectroscopy. The ESR spectra of the spin labeled steroid in the presence of AAG could be used to characterize the ligand-protein interaction at equilibrium without the need of a separation between bound and free species. An association constant Ka of 6.10(5) M-1 at 20 degrees C and a binding capacity of one site per mole protein were found. ESR spectra recorded at equilibrium at various temperatures allowed the calculation of enthalpy and entropy variations for the steroid-protein interaction; these thermodynamic parameters exhibited a rapid change above 45 degrees C which may be related to a protein conformational modification above this temperature, as detected by circular dichroism study. The ESR spectra width could be used to define a polar character for the spin label environment in the steroid binding site of AAG and to calculate an apparent rotational correlation time of 2.8 x 10(-8) sec for the steroid-protein complex in aqueous solution at 20 degrees C. It can be concluded that spin labeling and ESR methodology is of value in the study of steroid-protein interactions of biological significance above all because it can provide direct physico-chemical information concerning the local environment of the ligand in its binding site at equilibrium.     

Molecular dynamics in protein-single stranded DNA complexes. Two distinct nucleoside mobilities, in poly(deoxythymidylic acid)-poly-L-lysine complexes

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time tau -R = 2 ns, and the other one by a tau -R greater than or equal to 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Molecular Dynamics in Protein—Single Stranded DNA Complexes. Two Distinct Nucleoside Mobilities in Poly(deoxythymidylic acid)—poly-L-lysine Complexes

Abstract

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time τR = 2 ns, and the other one by a τR ≤ 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Synthesis and use of new spin labeled derivatives of phosphorylcholine in a comparative study of human, dogfish, and Limulus C-reactive proteins

New spin labeled derivatives of phosphorylcholine have been synthesized. The compounds cause reversible inhibition of the precipitation reactions between pneumococcal C-polysaccharide and the C-reactive proteins from humans, dogfish sharks (Mustelus canis), and horseshoe crabs (Limulus polyphemus). The spin labeled phosphorylcholine derivatives also rival phosphorylcholine as a ligand for the human, dogfish, and Limulus C-reactive proteins. The interactions of the purified C-reactive proteins with the spin labeled derivatives of phosphorylcholine have been studied using electron spin resonance spectrometry. The dramatic decrease in the ESR signal of some of the spin labels is due to immobilization of the label. Only the well known phosphate spin label, 4-phosphate-2,2,6,6-tetramethylpiperidine-1-oxyl could be used for binding studies on human and Limulus C-reactive proteins. Thus, by Scatchard analysis, the human C-reactive protein bound 1 mol of phosphate spin label per mol of protein with a Ka = 3.91 X 10(3) M-1, whereas the Limulus C-reactive protein bound only 0.5 mol of phosphate spin label per mol of protein with an overall Ka = 1.95 X 10(3) M-1. Inhibition studies using purified C-polysaccharide-induced inhibition of the phosphate spin label-human C-reactive protein interaction showed competitive inhibition with a KI of 4.78 X 10(-5) M at 18 degrees C. The phosphate spin label did not bind to dogfish C-reactive protein. However, one new phosphorylcholine spin label did bind and was used for Scatchard and Hill plot analyses. The dogfish C-reactive protein, which exists as a Mr = 50,000 dimer, bound 2 mol of the phosphorylcholine spin label per mol of protein, and this binding exhibited negative cooperativity as indicated by a Hill coefficient of 0.75.     

Interaction of albumin with the endothelial cell surface

Endothelial cells (EC) are covered with cell-borne proteoglycans and glycoproteins. Blood plasma proteins (e.g., albumin) adsorb to this glycocalyx forming a complex endothelial surface layer (ESL). We determined the molecular mobility of albumin by electron spin resonance (ESR) in the presence and absence of ECs to analyze interactions with the ESL. Albumin was spin labeled with 5- or 12-4,4-dimethyloxazolidine-N-oxyl (DOXYL)-stearic acid yielding information on the mobility of the molecular surface (5-DOXYL) or the entire protein (12-DOXYL). EC cultures grown on glass coverslips were immersed in labeled albumin and placed in the temperature-regulated cavity of an ESR spectrometer. Alternatively, ECs were labeled and then exposed to native albumin. At 37 degrees C, rotational correlation times determined by modified saturation transfer ESR (ST-ESR) were 26 and 48 ns for 5-DOXYL- and 12-DOXYL-labeled albumin, respectively. Presence of ECs increased rotational correlation time values for 5-DOXYL-stearic acid to 37 ns but not for 12-DOXYL-stearic acid. Albumin was able to completely take up the label from labeled EC within 2 min. The present study shows that modified ST-ESR can be used to determine the mobility of biological macromolecules interacting with cellular surfaces. Reduction in albumin surface mobility in the presence of EC at unchanged mobility of protein proper and fast removal of labeled fatty acids from EC membranes indicate rapid transient interactions between albumin surface and ESL but no rigid incorporation of albumin into a macromolecular network that would interfere with its transport function for poorly water-soluble substances.     

Conformational change in thrombospondin induced by removal of bound Ca2+. A spin label approach

The effect of removal of Ca2+ bound to thrombospondin (TSP) on the protein structure in solution has been investigated using ESR spin-label techniques. A maleimide spin label was selectively attached to the free thiol group presumably near the carboxyl-terminal domain in which Ca2+-binding sites are situated. The ESR spectra of spin-labeled TSP showed that the bound label undergoes a relatively fast rotational motion with an effective rotational correlation time in the nano-second time regimes. Removal of bound Ca2+ in TSP by dialyzing spin-labeled TSP from a Ca2+-containing buffer into an EDTA-containing buffer resulted in an increase in the mobility of the bound label by a factor of 2.3. The data suggest that EDTA chelation of bound Ca2+ in TSP induces a conformational change of TSP at least near the site of spin labeling.     

Spin-labeling studies of beef-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase was spin labeled with a nitroxide derivative of parachloromercuribenzoate. The ESR spectrum was of the immobilized type and the labeling yield 0.6 mole of spin label bound per mole of protomer under standard conditions. The specific activity of the labeled enzyme was not modified but the activation by ADP abolished. Inhibition by GTP was not altered but the ESR spectrum showed that the bound spin label was further immobilized in the presence of GTP and NADPH. In the presence of the coenzyme NADPH, the labeling yield decreased to half its initial value. Such a protection effect was observed neither with NADH nor with ADP.     

Evidence that the two free sulfhydryl groups of plasma fibronectin are in different local environments. Saturation-recovery electron spin resonance study.

Human plasma fibronectin is a dimer consisting of two subunits; each contains two cryptic thiol groups that were selectively labeled with an 15N,2H-maleimide spin label. Previous studies using conventional X-band electron spin resonance (ESR) methods showed that the spectrum of the labeled protein displays a single strongly immobilized component with an effective rotational correlation time of approximately 17 ns, suggesting that the physical environments of the two labeled sites per chain are indistinguishable. Here we have used saturation-recovery ESR to measure directly electron spin-lattice relaxation time (T1) of the labeled protein in solution at 27 degrees C. Interestingly, the time evolution of the signal was found to be biphasic, which was deconvoluted into two T1 values of 1.37 and 4.53 microseconds. Thus, the two spin-labeled sulfhydryl sites of plasma fibronectin (Fn), being similar in rates of rotational diffusion, differ by a factor of 3.2 in T1. Parallel experiments using various fibronectin fragments showed that the 1.37-microseconds component is associated with the label attached onto the thiol located in between the DNA-binding and the cell-binding domains, and the 4.53-microseconds component is associated with the label attached onto the thiol located within the carboxyl-terminal fibrin-binding domain. The data suggest that the saturation-recovery ESR is a useful method for differentiating multiple spin-labeled sites on macromolecules in which the labels undergo similar rates of rotational motion.     

Molecular dynamics in protein-single stranded DNA complexes. Two distinct nucleoside mobilities in poly(deoxythymidylic acid)-gene 5 protein complexes

A set of differently spin labeled (dT)n is used to evaluate thymidine dynamics and some of the structural features in a (dT)n-gene 5 protein complex. ESR evidence is presented that only one of the four thymidine residues bound in the DNA binding channel shows strong immobilization, whereas the other three display significant mobility of the order of nanoseconds. It is hypothesized that the accessability of such mobile bases could be critical to the recognition of the (dT)n-gene 5 protein complex in auxiliary interactions with other proteins and competitive DNAs.     

High frequency dynamics in hemoglobin measured by magnetic relaxation dispersion

The magnetic relaxation dispersion profiles for formate, acetate, and water protons are reported for aqueous solutions of hemoglobin singly and doubly labeled with a nitroxide and mercury(II) ion at cysteines at beta-93. Using two spin labels, one nuclear and one electron spin, a long intramolecular vector is defined between the two beta-93 positions in the protein. The paramagnetic contributions to the observed 1H spin-lattice relaxation rate constant are isolated from the magnetic relaxation dispersion profiles obtained on a dual-magnet apparatus that provides spectral density functions characterizing fluctuations sensed by intermoment dipolar interactions in the time range from the tens of microseconds to approximately 1 ps. Both formate and acetate ions are found to bind specifically within 5 angstroms of the beta-93 spin-label position and the relaxation dispersion has inflection points corresponding to correlation times of 30 ps and 4 ns for both ions. The 4-ns motion is identified with exchange of the anions from the site, whereas the 30-ps correlation time is identified with relative motions of the spin label and the bound anion in the protein environment close to beta-93. The magnetic field dependence of the paramagnetic contributions in both cases is well described by a simple Lorentzian spectral density function; no peaks in the spectral density function are observed. Therefore, the high frequency motions of the protein monitored by the intramolecular vector defined by the electron and nuclear spin are well characterized by a stationary random function of time. Attempts to examine long vector fluctuations by employing electron spin and nuclear spin double-labeling techniques did not yield unambiguous characterization of the high frequency motions of the vector between beta-93 positions on different chains.     

Structural analysis of nucleic acids by labeled oligonucleotides

In this report, the characterization of labeled oligonucleotides was discussed from the view points of base sequence analysis and structural analysis of nucleic acids in solution. Oligonucleotides site specifically spin labeled with TEMPO and fluorescent labeled with fluorescein were prepared and used for those analyses. The changes of ESR lines and rotational correlation time (tau) of the spin labeled oligonucleotide (S-probe) were dependent on the base sequence of S-probe, diastereoisomers, and the manner of hybridization. These results suggest that the conformation of the hybrid largely affected the local mobility of TEMPO and that tau value of S-probe reflected the local structure of the hybrid. When S-probe which was complementary to a single strand region of 5S RNA, was mixed with 5S RNA, tau value largely changed, indicating that the S-probe could form hybrid with 5S RNA in solution. Similar results were also obtained in the fluorescence depolarization analysis using fluorescent labeled oligonucleotide (F-probe). These results suggest that S-probe and F-probe are capable for the recognition of the secondary structure of 5S RNA in solution and useful for the analysis of the secondary structure of other nucleic acids in solution.     

Saturation transfer electron spin resonance of Ca2(+)-ATPase covalently spin-labeled with beta-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels.

The Ca2(+)-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide beta-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the alpha-carbon, to enhance further the electrophilic character of the beta-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2(+)-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the linewidths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer lineheight ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the lineheight ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the lineheight ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2(+)-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.     

Photoaffinity spin-labeling of the Ca2+ ATPase in sarcoplasmic reticulum : Evidence for oligomeric structure

A spin labeled fatty acid (16-doxylstearic acid) was linked to a photochemical reacting group (azido derivative). When the molecule is introduced, at a low concentration, into rabbit sarcoplasmic reticulum membranes, the spectrum before illumination is identical to the spectrum obtained with the corresponding spin labeled fatty acid. After illumination, a large immobilized components is seen. It corresponds to about 70% of the ESR signal of the effectively bound label, at room temperature. The fraction of immobilized component varies with temperature, from 100% at 0°C to 50% at 35°C. Addition of a small amount of detergent (dodecyl octaethylene glycol monoether), under non solubilizing conditions, decreases the fraction of signal due to a strongly immobilized probe. A possible interpretation is that the immobilized signal reflects protein bound spin labels trapped in Ca²⁺ ATPase oligomers, which are partially dissociated by detergent addition or temperature increase.     

Modification of human serum albumin with N-(2,5-dinitro-4-fluorophenyl)-4-amino-2,2,6,6-tetramethyl-piperidinooxy radical.

Human serum albumin has been treated with the spin-labeling reagent indicated in the title. Ultraviolet spectral studies of the protein so modified suggest that reaction takes place at lysine and tyrosine sidechains; kinetic experiments indicate that there are two especially reactive amino groups of the protein which are preferentially modified. Evidence is presented that these groups include the one acetylated by aspirin (Lys-199) or those arylated by 2.6-dinitro-4-trifluoromethylbenzenesulfonate. Esr experiments show that bound spin labels have about the same correlation time expected for overall tumbling of the protein; ESR observations indicate that molecular freedom near the spin labels is not increased when the protein is transferred to 8 M urea.     

Saturation transfer electron paramagnetic resonance on membrane bound proteins. II-Absence of rotational diffusion of the cholinergic receptor protein in Torpedo marmorata membrane fragments.

We have applied the technique of saturation transfer electron paramagnetic resonance to study the rotational diffusion of spin labeled membrane bound cholinergic receptors from Torpedo marmorata. Two different spin labels were used: a spin labeled maleimide derivative which binds covalently to proteins and a long chain spin labeled acylcholine which binds reversibly with a high affinity to the receptor protein. The maleimide spin label has a motion whose rotational correlation time is τ₂ > 10^?3 sec. The long chain spin labeled acylcholine indicates slightly more motion ($\text{τ}{}_2\text{? 10}{}^{\mathrm{?4}}\text{sec}$), but the nitroxide in this latter case is probably more loosely bound.     

Analysis of mobility of protein side chains by spin label technique

Five spin labeled derivatives of a neurotoxin from cobra venom were analyzed by the earlier suggested method. The procedure was adjusted to the complex motional behaviour of the label. Each protein derivative carried covalently bound spin label on different lysine residues. In two derivatives, at positions Lys44 and Lys46, the labels were strongly mobile, whereas for other three derivatives modified at Lys15, Lys25 and Lys26 the label was less mobile with respect to the protein molecule, which made possible determination of the rotational correlation time of the protein molecule (2.8±0.3 ns). The rotational correlation time was in good agreement with the calculated value for the rigid sphere of the corresponding molecular weight. On the basis of the estimate of the anisotropic motion degree, it was found from the order parameter S that the label mobility increases in the following series of lysine residues: Lys26, Lys25, Lys15, Lys46, and Lys44. From the analysis of positions of outer wide peaks in ESR spectra obtained by varying temperature and viscosity of the medium, we determined the parameters for computer simulation. The theoretical and experimental spectra were found to be in good agreement.Nomenclature rotational correlation time of the protein molecule - _l rotational correlation time of the spin label - 2A _Z , 2A the rigid limit distance between OWP and IWP, respectively, for ESR spectra of spin labeled proteins - 2, 2 the averaged limit distance between the OWP and IWP correspondingly mobile spin label to respect of protein moiety with; = - 2A',2A distance between OWP and IWP in the ESR spectra of spin labeled proteins for any T and media Abbreviations SL spin label - NT neurotoxin II from cobra venom - NT-SL-Lys44 neurotoxin spin labeled at Lys44 residue - OWP outer wide peaks in the immobilized ESR spectra - IWP inner wide peaks in the immobilized ESR spectra - WL the residual linewidth     

Study by the spin-label method of relaxation properties of protein kinase, its subunits and the catalytic subunit--histone H1 complex

Using the spin label method, the rotational relaxation in solution of adenosine 3',5'-monophosphate-dependent protein kinase and its subunits as well as the complexes of the enzyme with the substrate, histone H1, was studied. The rotational correlation time of the spin labeled macromolecules was measured on the basis of the quantitative estimation of the label mobility in relation to the protein globule. The holoenzyme molecule was found to be a rigid sphere. Whereas the complex of the globular catalytic subunit of the enzyme with a specific protein substrate, the spin labeled histone H1, appeared a flexible formation. The relaxation properties of the histone H1 molecule selectively labeled by the spin label in its globular part were investigated.     

NH2-terminal spin labeling of human hemoglobin--spin states and temperature dependence

In order to explore fully how ligand- and temperature-induced alterations in the spin states of heme iron are related to protein readjustments, the spin label 4-isothiocyanate (I) was covalently attached at beta-93 cysteines and at NH2-terminal valines of various heme-iron ligand forms of human hemoglobin. It was found that the mobility of NH2-terminally bound spin labels depends on the magnetic moment of the heme iron. There is a an approximately linear relationship between the magnetic moment of the heme iron and the mobility of NH2-terminally bound spin labels. In accordance with our previous results, the temperature dependence of ESR spectra of spin-labeled hemoglobin suggests the temperature-induced protein conformational change in those heme-iron ligand forms that are characterized by the equilibrium of the spin states of the heme iron. The conformational change was sensed at both spin-label-binding sites: at beta-93 cysteines and at NH2-terminal valines.     

Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides.

Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mM tert-butyl hydroperoxide in the presence of 200 microM equine myoglobin or to 100 mM hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.