Comparison of the active sites of atropinesterase and some serine proteases by spin-labeling

The side chain of the serine residue in the active center of atropinesterase (AtrE), alpha-chymotrypsin (Chymo), and subtilisin A (Sub) was labeled with two paramagnetic reporter groups of different size (label I or II, respectively) by sulfonylation with N-[3-(fluorosulfonyl)phenyl]-1-oxy-2,2,5,5-tetramethyl-pyrroline-3 -carboxamide or N-[6-(fluorosulfonyl)-2-naphthyl]-1-oxy-2,2,5,5-tetramethylpyrroline+ ++-3 -carboxamide. ESR spectra of labeled enzymes in 10 mM phosphate buffer, pH 7.4, were measured at temperatures between 133 and 298 K by using a home-built spectrometer operating in the absorption mode at 10-kHz field modulation. The spectra, in particular those at 276-298 K, were analyzed by computer simulation of the overall line shape according to the methods developed by Freed and co-workers, based on eigenfunction expansion. In the case of AtrE for both labels, the best agreement between experimental and simulated solution spectra was obtained with only one mobility component showing anisotropic, axially symmetric reorientation according to the Egelstaff jump-diffusion model. The axis of preferential reorientation was found to lie in the XZ plane at a polar angle of about 30 degrees with the X axis. The corresponding rotational correlation time (tau parallel) did not show appreciable viscosity/temperature (eta/T) dependence but had a constant value of 4.4 and 2.2 ns for labels I and II, respectively. The rotational correlation time associated with rotation around the axes perpendicular to that of preferential reorientation (tau perpendicular) showed the usual eta/T dependence and had a value of 22.0 ns at 276 K for both labels. The above results strongly suggest that in AtrE both nonpolar reporter groups reside in a pocket near the active serine. Contrary to the situation in AtrE, the overall mobility of the -N-O. fragments in Chymo and Sub was found to result from contributions of at least two distinct motional states, strongly and weakly immobilized. In going from label I to label II, the relative contribution of the latter state increases at the expense of that of the former. This is ascribed to an equilibrium between a relatively free state of the aromatic cores and a firmly bound position in the specificity pocket of these proteases. The apparently more rigid embedding of the spin-labels in the enzyme structure of AtrE suggests that the size of the nonpolar binding pocket in the active center region of this esterase allows a deeper penetration of the aromatic portions of the labels than is possible for the specificity pocket of Chymo or Sub.     

Comparison of the active-site conformations of bovine α-thrombin and meizothrombin(desF1) by electron spin resonance

The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labelsm-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl)-m-(fluorosulfonyl)benzamide] andm-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-m-(fluorosulfonyl)benzamide], which are sensitive to differences betweenα- andγ-thrombin, were quite similar to that ofα-thrombin. That is, no major conformational differences between meizothrombin(desF1) andα-thrombin were observed in this region of the extended active site. On the other hand,p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide],p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-(fluorosulfonyl)benzamide], andm-VII [N-[m-(fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl-piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility betweenα-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labelsp-IV,p-V, andm-VII, which corresponds to the apolar binding region, differs in conformation fromα-thrombin.     

Protein substrate binding induces conformational changes in the chaperonin GroEL. A suggested mechanism for unfoldase activity

Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.     

High-resolution probing of local conformational changes in proteins by the use of multiple labeling: unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes

Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)-ethyl)amino)naphtalene-1-sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation. HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 A from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 A from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At approximately 1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 A in diameter depending on the experimental conditions and spectroscopic technique used.     

Selective spin-labeling of the ribosomal proteins of 70S ribosomes from Escherichia coli.

We have used a series of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) maleimide spin labels of different length to label, covalently and selectively, the most reactive sulfhydryl groups of 70S ribosomal proteins of Escherichia coli. Under short periods of labeling (1--2 min), less than two spin labels per ribosome are incorporated and were shown to be distributed mainly on five ribosomal proteins in the following order: S18 greater than S21, L27 greater than S17, and S12. With a long period of labeling (3 h) up to 13 spin labels are attached to the ribosome, and protein S1 is the most labeled. The shape of the electron paramagnetic resonance (epr) signal shows two components with a predominance for the strongly immobilized orientation, and the percentage of these components in each spectra has been evaluated. When the distance between the nitroxide group and the maleimide-attaching group exceeds 6 A (1 A = 0.1 nm) the strongly immobilized orientation disappears. The effect of magnesium ions on these selectively spinlabeled ribosomes shows that the dissociation into subunits does not affect the epr signal, but more spin labels are incorporated into the subunits if labeling is performed under conditions of dissociation.     

Binding of spin-labeled carboxyatractylate to mitochondrial adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier as studied by electron spin resonance

The spin-label 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid was attached to the inhibitor carboxyatractylate of the mitochondrial ADP/ATP carrier. Being closely linked to the inhibitor, the spin-label should reflect the mobility of the carboxyatractylate. When bound to the carrier in mitochondria, spin-labeled carboxyatractylate reveals a most unusual hyperfine splitting of 72 G. A second spectral component with a hyperfine splitting of 62 G is also mainly due to carrier-bound inhibitor. A similar spectrum with somewhat reduced hyperfine splitting was observed with the detergent-solubilized protein, whereas reincorporation into phospholipid membranes yielded almost the same spectra as in mitochondria. The carrier-bound spin-label is concluded to be highly immobilized. The less immobilized spectral component is discussed in terms of strongly anisotropic label motion. In addition, the unusual splitting is interpreted to indicate the highly polar environment of the nitroxide. The interpretations are supported by the temperature dependence, which indicates a reversible progressive spin-label mobilization up to 50 degrees C. Membrane-impermeable reducing agents showed that the spin-label is easily accessible from the aqueous phase.     

Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor

Three nitroxide spin-labeled monoderivatives of bovine pancreatic trypsin inhibitor were prepared with the amino-specific reagent succinimidyl 1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-carboxylate. The monoderivatives were purified by ion-exchange and affinity chromatography. Thin-layer maps of tryptic peptides of the monoderivatives showed that the spin-label was incorporated at either the alpha-amino group, Lys-15, or Lys-26. Two-dimensional J-correlated 1H NMR spectra of the monoderivatives were recorded. Spectra were also recorded after reduction by ascorbic acid of the nitroxide label to hydroxylamine. With the nitroxide label present, significant line-broadening effects on many of the cross peaks in the spectra were observed. The extent of line broadening for the C alpha H-NH cross peaks was qualitatively correlated with the distance between the labeled amino group and the average C alpha H-NH position in the crystal structure. The spin-label affects cross peaks of protons within approximately 15 A. This study suggests that it is feasible to accumulate sufficient intramolecular distances in order to determine protein solution structures with the aid of distance geometry algorithms.     

Spin-label and fluorescence labeling studies of the thioester bonds in human alpha 2-macroglobulin

Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and the thiol-specific fluorescent probe 2-[(4-maleimidophenyl)amino]naphthalene-6-sulfonic acid sodium salt (MANS). The ESR and fluorescence results showed that these sulfhydryl group sites were at the base of a narrow crevice that is greater than or equal to 8 A deep. Although the bound MANS fluorophore was slightly blue shifted with an enhanced quantum yield vs the free label in water, the environment of the sulfhydryl site appeared to be of a polar nature when compared with the emission maxima in several solvents of varying polarity. The Glx residue participating in the thioester linkage in the intact protein was labeled with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl. The distance between the Glx and Cys moieties was estimated at greater than or equal to 10-25 A from double spin-labeling experiments.     

Mobility of spin-labelled sulfhydryl sites in excitable tissue

The spin-label 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl was shown to be attached to sulfhydryl groups of the walking leg nerve from the lobster Homarus americanus. Its ESR spectra indicated that it was in a highly immobilized environment. Removal of 90% of the phospholipid by chloroform-methanol extraction had no effect on the degree of immobilization. The ESR spectra of lipid extracted nerves or homogenized nerves showed marked increases in mobility of the spin label when subjected to urea, guanidine·HCl, pH, temperature, proteases, and a smaller shift in response to changes in monovalent cation concentrations. The results are interpreted as a protein conformational shift resulting in increased mobility of the spin-labelled site.     

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.     

Myosin head orientation and mobility during isometric contraction: effects of osmotic compression.

We have correlated the mobility and the generation of force of myosin heads by applying radial compression to isometrically contracting muscle fibers. Osmotic pressure was produced by dextran T-500, and its effect on the orientation and mobility of myosin heads labeled with N-(1-oxy-2,2,5,5-tetramethyl-4-pyperidinyl)maleimide was observed by conventional and saturation-transfer electron paramagnetic resonance methods. A biphasic behavior is spectral changes coinciding with the tension dependence was observed as the fibers were compressed. At diameters above the equilibrium spacing, the large myosin head disorder characteristic during contraction in the absence of compression was largely maintained, whereas the mobility decreased threefold, from tauR approximately 25 microseconds to approximately 80-90 microseconds. The inhibition of fast microsecond motions was not accompanied by tension loss, implying that these motions are not necessary for force generation. At diameters below the equilibrium spacing, both the disorder and the mobility decreased dramatically in parallel with the tension inhibition, suggesting that slower microsecond motions and the disorder of the myosin head are necessary for muscle function.     

Conformational changes at the active site of pantetheine hydrolase during denaturation by guanidine hydrochloride

Conformational changes at the active site of pantetheine hydrolase (EC3.5.1.-) during guanidine hydrochloride (GndHCl) denaturation were investigated by UV and circular dichroism spectroscopy and by electron spin resonance spectroscopy, following the spectral behaviour of the nitroxide radicals (N- (1- oxyl - 2,2,5,5, -tetramethyl-3-pyrrolidinyl) iodacetamide) covalently linked to the two active site cysteine residues. At low denaturant concentrations (0.2 M) no conformational changes may be observed, whereas the catalytic activity, is strongly affected. The results indicate that the active site of pantetheine hydrolase is labile and unfolds under conditions in which no global tertiary struscture modifications can be observed.     

Synthesis of a new 8-spin-labeled analog of adenosine 5'-phosphate and its interaction with AMP nucleosidase.

The synthesis of a new 8-spin-labeled analog of AMP, 8-[[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)carbamoyl]methyl]thio]adenosine 5'-phosphate (8-slAMP), is described. The procedure is facile and results in high yields. 8-slAMP is a competitive inhibitor of AMP nucleosidase with a Ki of 19 microM as compared to a Km of 100 microM for AMP. The analog is not a substrate for the enzyme and does not displace MgATP2- from the allosteric sites under the usual assay conditions. The EPR spectrum of the bound spin probe reveals a highly immobilized nitroxide group. Binding studies with 8-slAMP at 8 degrees C indicate three independent binding sites (Kd = 1.4 microM) per molecule of enzyme (Mr = 320,000). These properties make 8-slAMP a good spin probe for AMP nucleosidase. The analog may also be useful for other proteins known or suspected of binding AMP analogs in a syn conformation.     

Bovine serum albumin: characterization of a fatty acid binding site on the N-terminal peptic fragment using a new spin-label

A new spin-label, 4-(L-glutamo)-4'-[(1-oxy-2,2,5,5-tetramethyl-3L-pyrrolidinyl )amino]-3, 3'-dinitrodiphenyl sulfone, is shown to bind to one high-affinity binding site on bovine serum albumin (K = 5 X 10(4) M-1, n = 1). Analysis of the binding of the spin-label to the amino-terminal half (peptic fragment PB) and the carboxy-terminal half (peptic fragment PA) of BSA, and their complex (PA-PB), indicates that the spin-label binds to a long-chain fatty acid binding site located on PB. The usefulness of the novel specificity of the spin-label in characterizing this binding site is discussed.     

Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides

Spin labels have been extensively used to study the dynamics of oligonucleotides. Spin labels that are more rigidly attached to a base in an oligonucleotide experience much larger changes in their range of motion than those that are loosely tethered. Thus, their electron paramagnetic resonance spectra show larger changes in response to differences in the mobility of the oligonucleotides to which they are attached. An example of this is 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine (1). How ever, the synthesis of this modified DNA base is quite involved and, here, we report the synthesis of a new spin-labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (2). This spin label is readily prepared in half the number of steps required for 1, and yet behaves in a spectroscopically analogous manner to 1 in oligonucleotides. Finally, it is shown here that both spin labels 1 and 2 can be used to detect the formation of both double-stranded and triplex DNA.     

Study of ubiquinone binding in ubiquinol-cytochrome c reductase by spin labeled ubiquinone derivative

A functionally active, spin labeled ubiquinone derivative, 2,3-dimethoxy -5-methyl-6-{10-(2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl-3-carboxy)-decyl}-1,4-benzoquinone, has been synthesized for the study of ubiquinone binding in ubiquinol-cytochrome $\text{c}$ reductase. When this spin labeled ubiquinone derivative interacted with ubiquinone- and phospholipid-depleted reductase, the spin label was totally immobilized. However, when phospholipids were replenished, the spin label showed mobility behaviour similar to that observed in a hydrophobic environment, indicating that the alkyl side chain of ubiquinone is extended into the hydrophobic region of intact reductase and has some degree of mobility.     

Properties of spin and fluorescent labels at a receptor-ligand interface.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.     

Spin label studies on the interactions of bovine adrenodoxin with NADPH-adrenodoxin reductase and with cytochrome P-450scc.

Adrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)imidazole (I) and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While assition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum.     

Spin-labeled polyribonucleotides

Poly (U), poly (C) and poly (A) were spin labeled with N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)-imidazole. This spin label interacts selectively with 2' OH ribose groups of polynucleotides and does not modify the nucleic acid bases. The extent of spin labeling is not dependent upon the nature of the base and is entirely determined by rigidity of the secondary structure of the polynucleotide. The extent of modification for poly (U), poly (C) and poly (A) was 4.2, 1.7 and 1.5 per cent, respectively, the secondary structure of the polynucleotides being practically unchanged. Some physico-chemical properties of the spin-labeled polynucleotides were investigated by ESR spectroscopy. Rotational correlation times of the spin label and activation energy of its motion were calculated.