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.     

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.     

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.     

Calcium-dependent stabilization of the central sequence between Met(76) and Ser(81) in vertebrate calmodulin

Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met(76) and Ser(81) in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met(76) and Ser(81) in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met(76) and Asp(80) in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995, Nat. Struct. Biol. 2:768-776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met(76) and Ser(81) and modulates the structural coupling between the opposing domains of CaM.     

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (T_m) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer T_m between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the T_m for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.     

Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.     

Carbonylcyanide p-trifluoro-methoxyphenylhydrazone-induced change of mitochondrial membrane structure revealed by lipid and protein spin labeling.

Structural information on the phenomena accompanying uncoupling of oxidative phosphorylation in mitochondria was obtained using lipid and protein spin labels. The event of partitioning, observed with a small lipid spin label, the 4,4-dimethyl-2,2-dipentyl-oxazolidine-3-oxide (6-N-11) has been studied. The ratio of polar/hydrophobic part of the third line of the spectra was decreased in the presence of the uncoupler carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP), probably indicating a higher proportion of hydrophobic environment of the label. Protein spin labels have been employed to study mobilities and rate of reduction of the labels. A long-chain maleimide spin label, the 3-2-(2-maleimidoethoxy)ethylcarbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl, in the presence of carbonylcyanide-p-trifluoro-methoxyphenylhydrazone revealed decreases of mobility and of the rate of reduction. Large amplification of these effects was obtained with a short-chain maleimide spin label, the 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. With this spin label, the effect of the uncoupler could be traced down to a concentration of 0.05 μm. It is concluded that both membrane lipid and protein are changed simultaneously in the uncoupling event.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells.

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 +/- 1.14 . 10(5) and (2.2 +/- 1.2) . 10(5) spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethylmaleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Versatility of oxazolidine spin labels--a model study with acyl-alpha-chymotrypsin.

The p-nitrophenyl ester of N-oxyl-4',4'-dimethyl-3-oxazolidinebutyric acid was synthesized. The resonance spectrum of the acyl-alpha-chymotrypsin intermediate of this substrate was found to have more motional freedom at the enzyme active site as compared to the acyl-enzyme prepared from the p-nitrophenyl esters of 1-oxyl-2,2,5,5-tetramethyl-3-carboxypyrrolidine and 1-oxyl-2,2,6,6-tetramethyl-4-carboxy-1,2,5,6-tetrahydropyridine. The flexibility and the versatility of Keana's oxazolidine spin labels as covalent conformational probes is discussed.     

Protein compactness measured by fluorescence resonance energy transfer. Human carbonic anhydrase ii is considerably expanded by the interaction of GroEL

Nine single-cysteine mutants were labeled with 5-(2-iodoacetylaminoethylamino)naphthalene-1-sulfonic acid, an efficient acceptor of Trp fluorescence in fluorescence resonance energy transfer. The ratio between the fluorescence intensity of the 5-(2-acetylaminoethylamino)naphthalene-1-sulfonic acid (AEDANS) moiety excited at 295 nm (Trp absorption) and 350 nm (direct AEDANS absorption) was used to estimate the average distances between the seven Trp residues in human carbonic anhydrase II (HCA II) and the AEDANS label. Guanidine HCl denaturation of the HCA II variants was also performed to obtain a curve that reflected the compactness of the protein at various stages of the unfolding, which could serve as a scale of the expansion of the protein. This approach was developed in this study and was used to estimate the compactness of HCA II during heat denaturation and interaction with GroEL. It was shown that thermally induced unfolding of HCA II proceeded only to the molten globule state. Reaching this state was sufficient to allow HCA II to bind to GroEL, and the volume of the molten globule intermediate increased approximately 2.2-fold compared with that of the native state. GroEL-bound HCA II expands to a volume three to four times that of the native state (to approximately 117,000 A(3)), which correlates well with a stretched and loosened-up HCA II molecule in an enlarged GroEL cavity. Recently, we found that HCA II binding causes such an inflation of the GroEL molecule, and this probably represents the mechanism by which GroEL actively stretches its protein substrates apart (Hammarstr?m, P., Persson, M., Owenius, R., Lindgren, M., and Carlsson, U. (2000) J. Biol. Chem. 275, 22832-22838), thereby facilitating rearrangement of misfolded structure.     

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.     

Computation of nitroxide-nitroxide distances in spin-labeled DNA duplexes

Nanometer distances in nucleic acids can be measured by EPR using two 1-oxyl-2,2,5,5-tetramethylpyrroline radicals, with each label attached via a methylene group to a phosphorothioate-substituted backbone position as one of two phosphorothioate diastereomers (R(P) and S(P)). Correlating the internitroxide distance to the geometry of the parent molecule requires computational analysis of the label conformers. Here, we report sixteen 4-ns MD simulations on a DNA duplex d(CTACTGCTTTAG) .d(CTAAAGCAGTAG) with label pairs at C7/C19, T5/A17, and T2/T14, respectively. For each labeled duplex, four simulations were performed with S(P)/S(P), R(P)/R(P), S(P)/R(P), and R(P)/S(P) labels, with initial all trans label conformations. Another set of four simulations was performed for the 7/19-labeled duplex using a different label starting conformation. The average internitroxide distance r(MD) was within 0.2 A for the two sets of simulations for the 7/19-labeled duplex, indicating sufficient sampling of conformational space. For all three labeled duplexes studied, r(MD) agreed with experimental values, as well as with average distances obtained from an efficient conformer search algorithm (NASNOX). The simulations also showed that the labels have conformational preferences determined by the linker chemistry and label-DNA interactions. These results establish computational algorithms that allow use of the 1-oxyl-2,2,5,5-tetramethylpyrroline label for mapping global structures of nucleic acids.     

A spin label that binds to myosin heads in muscle fibers with its principal axis parallel to the fiber axis.

We have used an indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione), designated InVSL, to study the orientation of myosin heads in bundles of chemically skinned rabbit psoas muscle fibers, with electron paramagnetic resonance (EPR) spectroscopy. After reversible preblocking with 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), we were able to attach most of the spin label covalently and rigidly to either Cys 707 (SH1) or Cys 697 (SH2) on myosin heads. EPR spectra of labeled fibers contained substantial contributions from both oriented and disordered populations of spin labels. Similar spectra were obtained from fibers decorated with InVSL-labeled myosin heads (subfragment 1), indicating that virtually all the spin labels in labeled fibers are on the myosin head. We specifically labeled SH2 with InVSL after reversible preblocking of the SH1 sites with 1-fluoro-2,4-dinitrobenzene (FDNB), resulting in a spectrum that indicated only disordered spin labels. Therefore, the oriented and disordered populations correspond to labels on SH1 and SH2, respectively. The spectrum of SH2-bound labels was subtracted to produce a spectrum corresponding to SH1-bound labels, which was used for further analysis. For this corrected spectrum, the angle between the fiber axis and the principal axis of the spin label was fitted well by a Gaussian distribution centered at theta o = 11 +/- 1 degree, with a full width at half-maximum of delta theta = 15 +/- 2 degrees. The unique orientation of InVSL, with its principal axis almost parallel to the fiber axis, makes it complementary to spin labels previously studied in this system. This label can provide unambiguous information about axial rotations of myosin heads, since any axial rotation of the head must be reflected in the same axial rotation of the principal axis of the probe, thus changing the hyperfine splitting. Therefore, InVSL-labeled fibers have ideal properties needed for further exploration myosin head orientation and rotational motion in muscle.     

Low microwave-amplitude ESR spectroscopy: measuring spin-relaxation interactions of moderately immobilized spin labels in proteins

Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1)<0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T(2e), is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-I252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T(2e). We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.     

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.     

Synthesis of chromophoric, spin label enzyme substrates useful for cryoenzymology.

The spin label nitroxide derivative 3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oic acid has been synthesized and characterized by chemical methods. It is a useful intermediate in the preparation of a new class of chromophoric spin label substrates for enzyme studies, as shown by the synthesis of O-3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oyl-L-beta-phenyllactic acid, a specific ester substrate of bovine pancreatic carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2). Kinetic parameters of the esterolytic reaction are conveniently determined by UV spectrophotometric methods, and a reaction intermediate can be stabilized in fluid cryosolvent mixtures at subzero temperatures. Results are presented of preliminary electron spin resonance studies to demonstrate that structural relationships of the spin label substrate in a catalytically active configuration to active site residues can be determined for this low temperature-stabilized reaction intermediate. This substrate thus demonstrates the utility of this new class of spin label derivatives for characterization of enzyme reaction intermediates stabilized by cryoenzymologic techniques.     

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.     

ESR analysis of the FAD in bovine liver monoamine oxidase

Two hydrazine spin labels, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carbonyl ethyl hydrazine and 1-oxyl-2,2,6,6-tetramethylpiperidino-4-hydrazine, were synthesized as probes of the FAD binding site of monoamine oxidase. The reporter nitroxide moiety showed an ESR spectrum classified as partially immobilized which is indicative of FAD near the surface of the enzyme. Attempts to pick up flavin semiquinone or free radical intermediates during substrate oxidation with the spin traps 5,5-dimethyl-1-pyrroline-1-oxidase and phenyl-t-butylnitrone were not successful.