Solvent Perturbation Research Articles

Structural analysis of botulinum neurotoxin types A and E in aqueous and nonpolar solvents by Fourier transform infrared, second derivative UV absorption, and circular dichroic spectroscopies

Two pharmacologically similar but antigenetically distinct botulinum neurotoxins, types A and E with a 1000-fold difference in their toxicity, were examined for nonpolar solvent-induced changes in secondary structures and polypeptide foldings to understand their structural differences and their comparative responsiveness/susceptibility to solvent perturbation. Analysis of far UV circular dichroic spectra in aqueous buffer for types A and E neurotoxins yielded the following: the alpha-helix contents were 27 and 20%; the beta-sheets were 36 and 44%, the beta-turns were 6.0 and 0%, and the random coils were 31 and 36%, respectively. Fourier transform infrared spectra, obtained by using attenuated total reflection technique, indicated high content of alpha-helix and beta-pleated sheet structures for both neurotoxins as judged by strong bands at 1651 and 1633 cm-1 in the amide I frequency region and bands at 1314 and 1245 cm-1 in the amide III frequency region. The peak height ratio of 1314 and 1245 cm-1 bands, suggests that the type A neurotoxin has slightly higher alpha-helical content than the type E neurotoxin. These observations are consistent with the secondary structures estimated from far UV circular dichroic spectra. Fourier transform infrared spectra of the neurotoxins, exposed to methanol, showed sharp increases of the 1651 cm-1 band and a significant increase in the height of the 1314 cm-1 band, suggesting increases in the alpha-helical contents of the proteins. The changes were more in the type A than in the type E neurotoxin. The changes were reversible upon reexposure of the proteins to the aqueous buffer. Second derivative absorption spectroscopy demonstrated that methanol also induced changes in the degree of Tyr exposure to solvent. The results are discussed in terms of structural differences between the single and dichain neurotoxins and in terms of their mode of action.

Unfolding transitions of fibronectin and its domains. Stabilization and structural alteration of the N-terminal domain by heparin

Changes in the conformational state of human plasma fibronectin and several of its fragments were studied by fluorescence emission, intrinsic fluorescence polarization and c.d. spectroscopy under conditions of guanidinium chloride-and temperature-induced unfolding. Fragments were chosen to represent all three types of internal structural homology in the protein. Low concentration (less than 2 M) of guanidinium chloride induced a gradual transition in the intact protein that was not characteristic of any of the isolated domains, suggesting the presence of interdomain interactions within the protein. Intermediate concentrations of guanidinium chloride (2-3 M) and moderately elevated temperatures (55-60 degrees C) induced a highly co-operative structural transition in intact fibronectin that was attributable to the central 110 kDa cell-binding domain. High temperatures (greater than 60 degrees C) produced a gradual unfolding in the intact protein attributable to the 29 kDa N-terminal heparin-binding and 40 kDa collagen-binding domains. Binding of heparin to intact fibronectin and to its N-terminal fragment stabilized the proteins against thermal unfolding. This was reflected in increased delta H for the unfolding transitions of the heparin-bound N-terminal fragment, as well as decreased accessibility to solvent perturbants of internal chromophores in this fragment when bound to heparin. These results help to account for the biological efficacy of the interaction between the fibronectin N-terminal domain and heparin, despite its relatively low affinity.

Interactions of Electromagnetic Radiation with Electrolytes at High to Ultra-High Frequencies

Abstract At frequencies above one megahertz, the behaviour of electrolytic solutions changes from an ionic conductor to a lossy dielectric. High frequency conductance measurements of aqueous electrolytes (Na2SO4 and KCl) have been made by continuous wave and pulse admittance methods, in cells with immersed, shiny Pt electrodes to study this transition. Peak conductance frequencies as a function of concentration did not conform to the classical circuit representation of a resistance and capacitance. Measured phase angles indicate that relatively concentrated electrolytes (0.001 − 1 M) exhibit inductance due to electromagnetic and perhaps mass (inertial) effects. Modelling was possible with a constant value of the inductance, L, and capacitance, C, using a simple RLC equivalent circuit. Ion and solvent perturbations in high frequency electrical fields first are discussed in terms of a damped harmonic oscillator model, whose macroscopic response is self-consistent and equivalent mathematically to RLC network...

Plastocyanin cytochrome f interaction.

Spinach plastocyanin and turnip cytochrome f have been covalently linked by using a water-soluble carbodiimide to yield an adduct of the two proteins. The redox potential of cytochrome f in the adduct was shifted by -20 mV relative to that of free cytochrome f, while the redox potential of plastocyanin in the adduct was the same as that of free plastocyanin. Solvent perturbation studies showed the degree of heme exposure in the adduct to be less than in free cytochrome f, indicating that plastocyanin was linked in such a way as to bury the exposed heme edge. Small changes were also observed when the resonance Raman spectrum of the adduct was compared to that of free cytochrome f. The adduct was incapable of interacting with or donating electrons to photosystem I. Peptide mapping and sequencing studies revealed two sites of linkage between the two proteins. In one site of linkage, Asp-44 of plastocyanin is covalently linked to Lys-187 of cytochrome f. This represents the first identification of a group on cytochrome f that is involved in the interaction with plastocyanin. The other site of linkage involves Glu-59 and/or Glu-60 of plastocyanin to as yet unidentified amino groups on cytochrome f. Euglena cytochrome c-552 could also be covalently linked to turnip cytochrome f, although with a lower efficiency than spinach plastocyanin. In contrast, a variety of cyanobacterial cytochrome c-553's and a cyanobacterial plastocyanin could not be covalently linked to turnip cytochrome f.

A comparative picosecond spectroscopic study of the competitive triple fluorescence of aminosalicylates and benzanilides

Picosecond time-resolved absorption spectroscopy and fluorescence excitation and decay dynamics studies have unravelled the complexities of triple competitive fluorescence in the aminosalicylates and benzanilides. The fluorescences observed are F 1, the normal locally excited state emission S 1 (LE) → S 0; F′ 2, the proton-transfer tautomer emission S′ 1 (PT) → S′ 0 (PT); and F″ 2, the twisted intramolecular charge-transfer emission S″ 1(TICT) → S 0. The triple fluorescence was observed for p-dimethylaminosalicylic acid methyl ester and for benzanilide, in comparison with other aminosalicylates and benzanilides. Structure selection and solvent perturbation permitted the “tuning in” of F 1, F′ 2, and F″ 2 fluorescences individually and in combination.

On the Origin of the Positive Band in the Far-ultraviolet Circular Dichroic Spectrum of Fibronectin

The genesis of the positive bands in the far-ultraviolet circular dichroic spectra of human plasma fibronectin and its 31-kDa NH2-terminal heparin-binding fragment was studied. Spectra of ester derivatives of tyrosine, tryptophan, and phenylalanine and of model mixtures of these derivatives in which they are present in the same ratios as in the proteins indicate that all the aromatic side chains make substantial contributions to composite positive bands with maxima several nanometers below those of the proteins. In the presence of solvent perturbants such as polyethylene glycol and ethylene glycol, the bands of the model mixtures are red-shifted to the approximate positions they have in the spectra of the proteins. No additional red shift is seen with solvent perturbation of the proteins, suggesting that the conditions leading to this effect are satisfied within the proteins. A separate effect of solvent perturbation, an increase in amplitude of the positive band, occurs equally in solutions of free aromatic amino acid derivatives and in the proteins. This effect is used to estimate the relative accessibility of the chromophores of fibronectin and its fragment to different perturbants. The possible influence of protein secondary structure on the amplitude of the positive circular dichroic band is discussed.

Reversible unfolding of the gelatin-binding domain of fibronectin: structural stability in relation to function.

Fibronectin, a large multidomain glycoprotein, binds denatured collagen (gelatin) and mediates cell attachment and spreading on collagen-coated surfaces. Despite the high affinity, binding to gelatin is disrupted by relatively mild conditions. We have examined the effects of denaturants on the structure and function of a 42-kDa gelatin-binding fragment (GBF) isolated from chymotryptic and thermolytic digests of the parent protein. Application of linear gradients to GBF-loaded gelatin-agarose columns resulted in peak elution of the fragment at pH 5.2 or 10.2, at 0.4 M dimethylformamide, 0.9 M GdmCl, or 2.0 M urea, conditions far short of those required to induce structural changes detectable by fluorescence or circular dichroism. Solvent perturbation, fluorescence quenching, and chemical modification experiments indicate that about half of the 8 tryptophans, one-third of the 21 tyrosines, and all of the 9 lysine residues are solvent-exposed in the native protein and that 1 or more of the latter are directly involved in binding to gelatin, most likely through a hydrogen-bonding mechanism. Titration with GdmCl produced a single unfolding transition centered near 2.5 M GdmCl as monitored by changes in fluorescence and circular dichroism. This transition was fully reversible with complete recovery of structural parameters and gelatin binding. Treatment with disulfide reducing agents caused rapid irreversible changes in structure similar to those produced by GdmCl with concomitant loss of gelatin binding. Thus, tertiary and secondary structures are important for binding, but binding can be disrupted without perturbing those structures.

Polyethylene as a medium for eliminating the solvent perturbation in intramolecular proton transfer systems

Polyethylene was utilized as a suitable medium for proton transfer systems. A low-temperature fluorescence study of 3-hydroxyflavone was used as a probe of the quality of polyethylene. It was found that perturbations due to H-bonded impurities are largely suppressed in the polymer matrix as compared with other media. As an example we reinvestigated the phenomenon of dual fluorescence of 2,5-bis-(2-benzoxazolyl)-4-methoxyphenol. In this particular proton-transfer system, the intrinsic character of the energy barrier for excited-state proton transfer was confirmed.

Polyethylene as a medium for eliminating the solvent perturbation in intramolecular proton transfer systems

Polyethylene was utilized as a suitable medium for proton transfer systems. A low-temperature fluorescence study of 3-hydroxy-flavone was used as a probe of the quality of polyethylene. It was found that perturbations due to H-bonded impurities are largely suppressed in the polymer matrix as compared with other media. As an example we reinvestigated the phenomenon of dual fluorescence of 2,5-bis-(2-benzoxazolyl)-4-methoxyphenol. In this particular proton-transfer system, the intrinsic character of the energy barrier for excited-state proton transfer was confirmed.

Excited state dipole moments and polarizabilities of centrosymmetric and dimeric molecules. III. Model calculations for 1,8-diphenyl-1,3,5,7-octatetraene

A vibronic coupling model is used for the quantitative interpretation of both the 1 1B u ← 1 1A g absorption and the 2 1A g → 1 1A g fluorescence spectra of 1,8-diphenyl-1,3,5,7-octatetraene (DPO) in cyclohexane solution at 298 K in the absence and in the presence of an external electric field (electro-optical spectra). It is assumed that the coupling of the close lying 2 1A g and 1 1B u states is mainly due to static perturbations by the solvent environment and occurs via two totally symmetric CC and CC bond stretching vibrations. The solvent perturbations give rise to non-zero dipole moments of the vibronic excited states and influence the excited state polarizabilities. Both quantities strongly depend on the considered vibronic level. This implies that the electro-optical absorption band is not homogeneous with respect to the electro-optical properties of the underlying transitions. Some general aspects of the evaluation of heterogeneous electro-optical spectra are discussed for a two-band system. The long axis polarizability of the fluorescent state of DPO is determined by integral electro-optical emission measurements.

Solvent perturbations on the excited state symmetry of randomly oriented molecules by two-photon absorption

The polarization dependence of two-photon absorption is used to determine the excited state symmetry of solute molecules dissolved in room temperature liquids. Perturbations of excited state symmetry are interpreted as solvent induced state mixing in the solute. Using a first-order expansion in the zero-order system wave functions, an expression is derived which describes the effects of solute–solvent electric dipole interactions on the symmetry of the solute excited state. Measurements on the S0→S1 0–0 band of fluorene in nonassociating polar and nonpolar liquids are fit well by the derived expression, using continuum reaction field models. Deviations from the derived expression are found when fluorene is dissolved in liquids capable of self-association or complexation with aromatic solutes. In n-alcohol solvents, perturbations on the excited state symmetry of fluorene are adequately accounted for using Kirkwood g factors for the solvent.

Excited state dipole moments and polarizabilities of centrosymmetric and dimeric molecules. II. Polyenes, polyynes and cumulenes

Electro-optical absorption spectra are measured for a series of polyenes, polyynes and cumulenes with centrosymmetric π-chromophores in cyclohexane solution at 298 K. For all molecules the long-axis component of the polarizability tensor is considerably larger in the first dipole-allowed singlet state compared to the ground state. The transition moments are found to be parallel to the long molecular axis. All polyenes and one cumulene show a linear Stark component indicating a long-axis excited state dipole moment. Both the dipole moments and the polarizabilities are corrected within the extended Onsager model for solvent cavity and reaction field effects. It is suggested that symmetry lowering solvent perturbations are the reason for the apparent excited state dipole moments.

States and roles of tryptosphan residues in Abrus precatorius agglutinin.

The states of tryptophan residues in Abrus precatorius agglutinin (APA) were analyzed by chemical modification and solvent perturbation UV-difference spectroscopy. The number of tryptophan residues available for N-bromosuccinimide (NBS) oxidation increased with lowering pH, and 20 out of the 24 tryptophans in APA were modified at pH 3.0, while 2 tryptophans were eventually oxidized at pH 5.0. Modification of tryptophan greatly decreased the binding of APA with saccharides, and only 4% of the hemagglutinating activity was retained after modification of 4 tryptophan residues/molecule. When the modification was done in the presence of lactose or galactose, 2 tryptophan residues/molecule remained unmodified with a retention of a fairly high hemagglutinating activity. The data from solvent perturbation UY-difference spectroscopy indicated that 6 tryptophans were on the surface of the APA molecule, and 4 tryptophan residues/molecule were shielded from the perturbing effect of the solvent upon binding with lactose. Based on these results, we proposed that in the saccharide-binding site on each B-chain of APA there exists one tryptophan residue directly involved in saccharide binding, and near the binding site there is another tryptophan residue whose state is also changeable upon binding with saccharide.

Resolution of L a and L b bands in methyl indoles by two-photon spectroscopy

Polarized two-photon fluorescence excitation spectra were obtained for indole, 5-methylindole, 3-methylindole and 2,3-dimeth-ylindole in cyclohexane and butanol solutions. The polarization ratios clearly indicate the onset of L a absorption and directly verify established models regarding the relative response of L a and L b state energies to substitution and solvent perturbations (prior to excited-state relaxations). The two-photon properties are successfully modeled by INDO/S CI computation.

The structure and internal rotational barrier of 3-phenyl-1-propyne by molecular orbital calculations and the J method

The 1H nuclear magnetic resonance spectral parameters are reported for 3-phenyl-1-propyne dissolved in CCl4, C6D6, and in acetone-d6. The long-range spin–spin coupling constants imply very small and perhaps vanishing barriers to internal rotation about the [Formula: see text] bond in all three solutions, in contrast to benzyl cyanide in which there exist significant solvent perturbations of the internal barrier. STO 3G MO computations, utilizing geometry optimization procedures, imply an internal rotational potential of V/kJ mol−1 = −2.8 sin2 ψ − 0.6 sin2 2ψ; the angle ψ is 90° when the C≡C bond lies in a plane perpendicular to the benzene plane. 6-31G MO energies imply V/kJ mol−1 = −0.3 sin2 ψ − 0.4 sin2 2ψ, the fourfold component being larger than the twofold. A flat minimum occurs near ψ = 50°.

Tryptophan fluorescence studies of subunit interaction and rotational dynamics of human luteinizing hormone.

Human luteinizing hormone (hLH) has a single tryptophan residue occurring in the beta-subunit (beta hLH). This provides an intrinsic fluorescent probe, in native hLH and beta hLH, that is unambiguously assigned. The fluorescence intensities of hLH and beta hLH are, however, significantly different. This difference has been utilized in studying the interaction of fluorescent beta hLH with the nonfluorescent alpha-subunit. The accessibility of the tryptophan residue in native hLH and beta hLH has been assessed by measuring the rate of collisional fluorescence quenching and by solvent perturbation (D2O/H2O) of fluorescence. Fluorescence anisotropy measurements have been used in studying the intramolecular dynamics and segmental tryptophan mobility in hLH and beta hLH. Lifetime-resolved anisotropy, measured by the technique of oxygen quenching of fluorescence, has revealed the presence of segmental tryptophan motion. These data can be satisfactorily explained in terms of fast segmental tryptophan motion and rotational diffusion of the whole protein and do not require that intersubunit motion be invoked for intact hLH as it was suggested earlier on the basis of fluorescence depolarization of fluorescein-labeled hLH [Bishop, W. H., & Ryan, R. J. (1975) Biochem. Biophys. Res. Commun. 65, 1184-1190].

The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent 'bait' region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

States of tryptophan residues in castor bean hemagglutinin: the perturbing effects of solvents on tryptophans in hemagglutinin and its complexes as analyzed by UV-difference spectroscopy.

The states of tryptophan residues in castor bean hemagglutinin (CBH) were analyzed by solvent perturbation studies employing ultraviolet difference spectroscopy. Eight out of 22 tryptophan residues in CBH were exposed to ethylene glycol and glycerol, suggesting that the remaining 14 tryptophan residues are buried in the interior of the CBH molecule. The fraction of tryptophan residues accessible to the perturbant decreased with increase in the molecular size of the perturbant, and only 2 tryptophan residues were exposed to polyethylene glycol 600. Upon binding with raffinose, 2 tryptophan residues were shielded from the perturbing effect of the solvent, and binding of lactose reduced the number of tryptophan residues accessible to the perturbant by 1 mol per mol of protein. Binding of galactose, however, did not change the accessibility of tryptophan to the perturbant. On the other hand, the accessibility of tyrosine to the perturbant remained unchanged after binding with raffinose and lactose, suggesting that tyrosine is not directly involved in the saccharide binding of CBH. Based on these results, it is proposed that one tryptophan residue at the saccharide-binding site on each B-chain of CBH lies on the surface of the protein molecule and is located at a subsite which is accessible to a glucopyranoside moiety in the lactose molecule or a glycopyranosyl-fructofuranosyl moiety in the raffinose molecule, whereas such a residue is not present at the galactopyranoside-recognition site.

Biophysical studies on the calcium trigger of muscle contraction.

The basic structural organization of striated muscle tissue consists of two types of filaments—thick and thin—which are interdigitated and slide past one another when muscle contracts. In the thick filaments the long matchsticklike myosin molecules are organized into a biopolar structure in which the tails form the core of the filament and the heads project out at both ends. The ATPase activity and acitin-binding properties are located in the projecting heads. It is the interaction of these heads with the thin filaments and the concomitant hydrolysis of ATP that provides the driving force for the sliding of thin over thick filaments. The core of the thin-filament structure is made up of a long double-stranded helical assembly of globular (G) acting monomers known as “F-actin.” Located in each of the two grooves of the F-actin structure is a filament of rodlike tropomyosin molecules aggregated head to tail and spanning the length of the thin filament. Each tropomyosin molecule spans seven G-actin monomers on each of the two strands of F-actin and interacts with one troponin complex. The latter consists of three proteins: troponin-C (TN-C), which binds calcium; troponin-I (TN-I), the inhibitory protein; and troponin-T (TN-T), which binds the troponin complex to tropomyosin. When a nerve impulse stimulates a muscle to contract there is an increase in the Ca2+ concentration in the fluid bathing the thick and thin filaments. Binding of this Ca2+ to the TN-C component triggers a series of conformational transitions in the troponin complex and a change in the position of tropomyosin from a blocking position in the grooves of F-actin. Myosin heads are then able to interact with F-actin and sliding of thick over thin filaments ensues. The research in our laboratory has been directed toward a fuller understanding of the detailed molecular mechanisms by which this intricate and sophisticated system operates in both skeletal and cardiac muscle. To this end we have explored the calcium-induced conformational change in TN-C and its propagation through the entire troponin–tropomyosin complex, using a combination of hydrodynamic (ultracentrifuge, viscosity, magnetic densitometry) and spectroscopic (CD, UV absorption difference, solvent perturbation difference, nmr, and fluorescence) approaches. Our studies to date have included the following: the development of highly purified troponin subunits, using high-performance liquid chromatography methodologies; the rigorous physicochemical characterization of the troponin subunits and their interaction properties; precise details of several of the functional groups (carboxyls, aromatics) involved in the Ca2+-induced conformational change in TN-C; the role of sulfhydryl groups in generating functionally active and conformationally sensitive troponin complexes; the molecular morphology of the troponin complex on the thin filament; the role of the two Ca2+-specific regulatory sites in TN-C in terms of their responsiveness to rapid Ca2+ transients; and the demonstration of a common regulatory mechanism for both skeletal and cardiac muscle, using bioassay and spectroscopic studies on ternary complexes made from hybrid subunits of both muscle types. The highlights of these studies are described, including the recently elucidated x-ray structure of TN-C and how the binding of calcium to such a structure may act as a conformational switch.

Solvent perturbation spectra of yellow mealworm α-amylase and of wheat protein inhibitors

1. 1. The number of tyrosine and tryptophan residue-equivalents on the surfaces of the α-amylase and of two of its protein inhibitors has been determined. 2. 2. The solvent-exposed tyrosine and tryptophan residue-equivalents in the dimeric inhibitor are respectively four and two times as much as those ones of the monomeric inhibitor. 3. 3. On the basis of the homology among their polypeptide chains, it is suggested that the outer tryptophan residues in either inhibitors are located at the positions 4 and 51 of the primary structure. 4. 4. On the interaction of the monomeric inhibitor with the amylase, two tryptophan residue-equivalents became no more accessible to the solvent.