Gd3+ vibronic side band spectroscopy. New optical probe of Ca2+ binding sites applied to biological macromolecules.

A new spectroscopic technique is presented for obtaining infraredlike spectra of the binding sites of Ca2+ and other metals in biological macromolecules. The technique, based on the Ca(2+)-like binding properties of Gd3+, utilizes vibronic side bands (VSB) that appear in Gd3+ fluorescence. In the fluorescence spectrum of Gd3+, the separation in photon frequency between a VSB and its electronic origin at approximately 32,150 cm-1 (approximately 311 nm) is a direct measure of the vibrational frequency of a ligand coordinated to Gd3+ ion. As a consequence, the VSB are uncomplicated by molecular vibrations distant from the Gd3+ binding site. The vibrational spectra resulting from the VSB of Gd3+ coordinated to a Ca2+ binding protein, a phospholipid, and DNA are presented.     

Near-infrared Yb(3+) vibronic sideband spectroscopy: application to Ca(2+)-binding proteins.

We have used near-infrared (NIR) vibronic fluorescence spectroscopy to study the vibrational structure of ligands associated with model complexes of the lanthanide Yb(3+). This technique exploits the similar binding properties of the lanthanide Yb(3+) to probe Ca(2+)-binding sites in proteins. The (NIR) fluorescence of complexed Yb(3+) exhibits, in addition to main 0-0 (2F5/2----2F7/2) electronic transition of Yb(3+), weak vibronic sidebands which provide infrared-like, local vibrational spectra of the chelates (inner sphere ligands) of Yb(3+). A similar approach has been used for the lanthanide Gd(3+) (MacGregor, R.B., Jr (1989) Arch. Biochem. Biophys. 274, 312-316) which fluoresces in the UV and which is usually complicated by amino-acid residues fluorescing in the same spectral region. In this same spectral region, other complications in studying photosynthetic membranes occur in the form of the excitation wavelength being actinic, promoting photodegradation of the membranes, as well as the reabsorption of Gd(3+) fluorescence. NIR excitation and fluorescence detection of Yb(3+) avoid these problems when studying photosynthetic membranes. A preliminary study has been conducted here on rat muscle parvalbumin.     

Intensity of cooperative vibronic transitions in the Eu(III), Gd(III) and Tb(III) formates spectra

The luminescence and absorption spectra of the lanthanide ions in solids and coordination compounds are characterized by sharp pure electronic lines, which are accompanied by much weaker lines of vibronic transitions. The vibronic spectroscopy is a good probing tool for investigations of the properties of surrounding ion ligands. The lanthanides formates are efficient luminescent crystals and can be viewed as the elementary type in the whole class of the oxygen-containing lanthanide coordination compounds. The intensity of vibronic transitions in spectra of luminescence and excitation europium (⁵D₀→⁷F₂, ⁷F₀→⁵D₂), terbium (⁷F₆→⁵D₄), gadolinium (⁶P_7/2→⁸S_7/2) in anhydrous formates of the type Ln(HCOO)₃ (Ln = Eu, Tb, Gd) and Y(HCOO)₃.2H₂O doped with Eu³⁺ and Tb³⁺ (C ~1 mol%) are reported. Also, the infrared and Raman spectra were obtained for the same compounds. Related integral intensity vibronic sidebands depend on the type of electronic transition of the same ion and varies for the same electronic transitions in different crystals. The obtained experimental data referring to the rate constants of vibronic transitions and intensity distribution in vibronic spectra on normal vibrations of the formate groups are in agreement with the predictions based on the Stavola–Dexter theory of cooperative vibronic transitions.     

Aromatic substrate specificity of horseradish peroxidase C studied by a combined fluorescence line narrowing/energy minimization approach: the effect of localized side-chain reorganization

Horseradish peroxidase C binds a wide variety of small H-donor compounds such as benzohydroxamic acid (BHA) and 2-naphthohydroxamic acid (NHA). In this work, we use the Mg(II)-mesoporphyrin prosthetic group derivative as a spectroscopic probe of the active site and of the interaction with the substrates. We report on high-resolution fluorescence line-narrowed spectra which show that the effects of substrate binding on the electronic transitions are similar for both substrates and present data on the normal vibrational modes that are active in the vibronic spectra. Analysis of the vibrational frequencies shows that the Mg(II) ion is 5-coordinate in all cases, thus ruling out a solvent water as sixth ligand. The frequency shifts observed as a result of substrate binding are also indicative of a more rigid prosthetic group upon substrate binding. We present models for MgMP-HRP and its complexes with both substrates and compare the resulting structures on the basis of a modeling approach combining energy minimization to finite difference Poisson--Boltzmann calculations which partitions the various relative protein contributions to substrate binding. We show that the electrostatic potential of the prosthetic group is modified by the binding event. Analysis of the unbound and bound energy-minimized structures shows that the enzyme modulates substrate binding by subtle charge reorganization in the vicinity of the catalytic site and that this rearrangement is not attributable to significant secondary structure conformational changes but to side-chain reorganization.     

Premelting and the hydrogen-exchange open state in synthetic RNA duplexes

Here we attempt to relate equilibrium temperature-dependent spectral changes in two synthetic RNA homopolymer duplexes—poly(rA) · poly(rU) and poly(rI) · poly(rC)—to the conformational opening detected in stopped-flow hydrogen–deuterium exchange experiments on these molecules. We are concerned with changes in several spectral properties that occur well below onset of the thermally induced helix-coil “melting” transition in these systems. These are known as “premelting” transitions, and can be detected in uv CD spectra as well as in vibrational bands of the bases in the ir. Both CD and ir spectra exhibit isoelliptic or isosbestic points consistent with a well-defined two-state premelting process. Application of a least-squares analysis to two-state models for premelting using data from different bands in the CD and ir shows that the enthalpies are substantially greater than that of the hydrogen-exchange opening. Thus the hydrogen-exchange open state represents only one premelting reaction among several that lead to equilibrium changes in helix geometry or base vibrational modes. The latter include processes that occur on a rapid time scale, including potential base-pair openings not productive for the exchange reaction. It appears that the former, and not the hydrogen-exchange opening, dominates the premelting alterations monitored by ir and CD spectroscopy.     

The determination of the absolute configurations of chiral molecules using vibrational circular dichroism (VCD) spectroscopy

The vibrational circular dichroism (VCD) spectra of the two enantiomers of a chiral molecule are of equal magnitude and opposite sign: i.e. mirror-image enantiomers give mirror-image VCD spectra. In principle, the absolute configuration (AC) of a chiral molecule can therefore be determined from its VCD spectrum. In practice, the determination of the AC of a chiral molecule from its experimental VCD spectrum requires a methodology which reliably predicts the VCD spectra of its enantiomers. The only reliable methodology developed to date uses the Stephens quantum-mechanical theory of the rotational strengths of fundamental vibrational transitions, developed in the early 1980s, implemented using ab initio density functional theory in the GAUSSIAN program in the mid 1990s. This methodology has by now been widely used in determining ACs from experimental VCD spectra. In this article we discuss the protocol for determining the ACs of chiral molecules with optimum reliability and its implementation for a variety of molecules, including the D3 symmetry perhydrotriphenylene, a thiazino-oxadiazolone recently shown to be a highly active calcium entry channel blocker, the alkaloid natural products schizozygine, iso-schizogaline, and iso-schizogamine, and the iridoid natural products plumericin, iso-plumericin, and prismatomerin. The power of VCD spectroscopy in determining ACs, even for large organic molecules and for substantially conformationally-flexible organic molecules is clearly documented.     

Gas-phase IR spectra of substituted alkylamides

The source of the splittings observed in the amide A, I, II, and III absorption bands of N-methylacetamide (NMA) have been determined by high-resolution gas-phase FTIR. Analysis of isotopically labeled NMA, along with simpler amides (formamide, acetamide) show that the observed splitting arise from rotational transitions within the vibrational manifold. The ir spectra of a homologous series of monosubstituted alkylamides are also examined. The observed effects on the ir spectra from incorporating substituents from methyl to t-butyl on both sides of the amide linkage are discussed in terms of rotational transitions and coupled oscillations. During these studies we did not observe any evidence for the presence of cis conformations of the amide bond.     

A novel spectroscopic probe for molecular chirality

Recent advances in developing sum frequency generation (SFG) as a novel spectroscopic probe for molecular chirality are reviewed. The basic principle underlying the technique is briefly described, in comparison with circular dichroism (CD). The significantly better sensitivity of the technique than CD is pointed out, and the reason is discussed. Bi-naphthol (BN) and amino acids are used as representatives for two different types of chiral molecules; the measured chirality in their electronic transitions can be understood by two different molecular models, respectively, that are extensions of models developed earlier for CD. Optically active or chiral SFG from vibrational transitions are weaker, but with the help of electronic-vibrational double resonance, the vibrational spectrum of a monolayer of BN has been obtained. Generally, optically active SFG is sufficiently sensitive to be employed to probe in-situ chirality of chiral monolayers and thin films.     

Effect of water molecules on the fluorescence enhancement of Aflatoxin B1 mediated by Aflatoxin B1:beta-cyclodextrin complexes. A theoretical study.

In order to explain the observed fluorescence enhancement of Aflatoxin B1 (AFB1) when forming AFB1:beta-cyclodextrin (AFB1:beta-CD) inclusion complexes, we have performed a theoretical (quantum chemistry calculations) study of AFB1 and AFB1:beta-CD in vacuum and in the presence of aqueous solvent. The AM1 method was used to calculate the absorption and emission wavelengths of these molecules. With the help of density functional theory (DFT) and time-dependent DFT (TDDFT) vibrational frequencies and related excitation energies of AFB1 and AFB1.(H2O)m = 4,5,6,11 were calculated. On the basis of these calculations we propose a plausible mechanism for the fluorescence enhancement of AFB1 in the presence of beta-CD: (1) before photoexcitation of AFB1 to its S1 excited state, there is a vibrational coupling between the vibrational modes involving the AFB1 carbonyl groups and the bending modes of the nearby water molecules (CG + WM); (2) these interactions allow a thermal relaxation of the excited AFB1 molecules that results in fluorescence quenching; (3) when the AFB1 molecules form inclusion complexes with beta-CD the CG + WM interaction decreases; and (4) this gives rise to a fluorescence enhancement.     

Vibrational CD of polypeptides. XII. Reevaluation of the Fourier transform vibrational CD of poly(gamma-benzyl-L-glutamate)

Using a newly constructed Fourier transform ir (FTIR) based vibrational CD (VCD) instrument, we have found that elimination of the ellipsoidal collection mirror before the detector and its replacement by a lens leads to a significant improvement in the absorption artifact problem often seen previously in FTIR-VCD. Reduction in artifact level brings the FTIR-VCD band shape of the amide 1 band in poly(γ-benzyl-L -glutamate) into better agreement with that found with dispersive instruments. These results indicate that the difference between the spectra obtained with the two methods is not primarily a factor of resolution but is more dependent on artifact level. Thus the previously reported deconvolution of the amide I VCD results and their subsequent interpretation in terms of the helical vs. exciton mechanisms overemphasize weak features in the spectrum. Results based on deconvolution of the absorption spectrum remain valid. Further new data on the amide II and several lower energy transitions that encompass the amide III region show broad single-signed features—but of the opposite sense—in the two regions, implying that their VCD arises through mutual coupling.     

A spectral hole-burning study of long-wavelength chlorophyll a forms in greening leaves at 5 K

Persistent holes have been burnt in the spectra of post-etiolated maize leaves in the spectral interval 700–745 nm at 5 K. Narrow (< 1 cm⁻¹) holes detectable both in fluorescence and excitation spectra correspond to the zero-phonon purely electronic S₁S₀ transition of chlorophyll a rather than to the S₁←S₀ vibronic or S₁←S₀ higher singlet transitions. The spectral composition of the inhomogeneously broadened absorption and fluorescence contours of long-wavelength chlorophyll a forms in plants is discussed in terms of the contributions of phonon-wings, vibronic satellites, purely electronic S₁S₀ transitions and its higher excitonic components.     

Toward a general mixed quantum/classical method for the calculation of the vibronic ECD of a flexible dye molecule with different stable conformers: Revisiting the case of 2,2,2‐trifluoro‐anthrylethanol

We extend a recently proposed mixed quantum/classical method for computing the vibronic electronic circular dichroism (ECD) spectrum of molecules with different conformers, to cases where more than one hindered rotation is present. The method generalizes the standard procedure, based on the simple Boltzmann average of the vibronic spectra of the stable conformers, and includes the contribution of structures that sample all the accessible conformational space. It is applied to the simulation of the ECD spectrum of (S)‐2,2,2‐trifluoroanthrylethanol, a molecule with easily interconvertible conformers, whose spectrum exhibits a pattern of alternating positive and negative vibronic peaks. Results are in very good agreement with experiment and show that spectra averaged over all the sampled conformational space can deviate significantly from the simple average of the contributions of the stable conformers. The present mixed quantum/classical method is able to capture the effect of the nonlinear dependence of the rotatory strength on the molecular structure and of the anharmonic couplings among the modes responsible for molecular flexibility. Despite its computational cost, the procedure is still affordable and promises to be useful in all cases where the ECD shape arises from a subtle balance between vibronic effects and conformational variety.     

Excited-State Lifetimes of Far-Infrared Collective Modes in Proteins

Vibrational excitations of low frequency collective modes are essential for functionally important conformational transitions in proteins. Here we report the first direct measurement on the lifetime of vibrational excitations of the collective modes at 87 pm (115 cm^-1) in bacteriorhodopsin, a transmembrane protein. The data show that these modes have extremely long lifetime of vibrational excitations, over 500 picoseconds, accommodating 1500vibrations. We suggest that there is a connection between this relativelyslow anharmonic relaxation rate of approximately 10 g sec^-1 and thesimilar observed rate of conformational transitions in proteins, which require require multi-level vibrational excitations and energy exchanges with othervibrational modes and collisional motions of solvent molecules.     

Resonance Raman spectra of copper(II)-substituted liver alcohol dehydrogenase: a type 1 copper analogue

Liver alcohol dehydrogenase (LADH) with copper in place of the catalytic zinc has recently been proposed to contain a type 1 site analogous to that in "blue" copper proteins. Resonance Raman spectra for the copper-substituted enzyme, Cu(II) X LADH, and its binary complexes with reduced nicotinamide adenine dinucleotide (NADH) and pyrazole support this viewpoint. These spectra have two dominant features: a sharp peak at approximately 415 cm-1, which is believed to be associated with vibration of the single histidine ligand, and a broader, asymmetric band at approximately 350 cm-1, whose components are assigned predominantly to vibrational modes of the two cysteinate ligands. The high frequency of these transitions, which is reminiscent of the blue copper proteins, is ascribed to the tetrahedral nature of the metal site that produces unusually short Cu-S bonds and coupled vibrational modes. Solvent exchange with H218O reveals no contribution to the resonance Raman spectrum of the water molecule, which is a metal ligand in free Cu(II) X LADH; however, the spectrum of the binary complex with pyrazole has several new peaks attributable, in part, to pyrazole ligation. The strong similarity among the vibrational spectra demonstrates that the Cu(II) environment in alcohol dehydrogenase maintains its near-tetrahedral geometry in the various enzyme derivatives. The resonance Raman spectrum of Ni(II) X LADH is close to that of Cu(II) X LADH and suggests a similar tetrahedral site. The Raman spectra presented here together with available optical and EPR data indicate that Cu(II) X LADH belongs to the type 1 copper classification and, thus, can provide new insights into this unusual coordination geometry.     

Heme symmetry, vibronic structure, and dynamics in heme proteins: ferrous nicotinate horse myoglobin and soybean leghemoglobin

We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg-Teller approximation. This theoretical approach makes use of the crude Born-Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands for both Condon and non-Condon vibrational modes. In this framework, an x-y splitting of the Q transition for native and deuteroheme reconstituted horse myoglobin is clearly assessed and attributed to electronic perturbations that, in turn, are caused by a reduction of the typical D(4h) symmetry of the system due to heme distortions of B(1g)-type symmetry and/or to an x-y asymmetric position of the nicotinate ring; in deuteroheme reconstituted horse myoglobin the asymmetric heme peripheral substituents add to the above effect(s). On the contrary, in leghemoglobin-a no spectral splitting upon nicotinate binding is observed, pointing to a planar heme configuration in which only distortions of A(1g)-type symmetry are effective and to which the nicotinate ring is bound in an x - y symmetric position. The local dynamic properties of the heme pocket of the three proteins are investigated through the temperature dependence of spectral line broadening. Leghemoglobin-a behaves as a softer matrix with respect to horse myoglobin, thus validating the hypothesis of a looser heme pocket conformation in the former protein, which allows a nondistorted heme configuration and a symmetric binding of the bulky nicotinate ligand.     

Analysis of the binding sites of Gd3+ on Ca(2+)-transporting ATPase of the sarcoplasmic reticulum through its effects on fluorescence of tryptophan residues and a covalently attached fluorescent probe N-(1-anilinonaphthyl-4)maleimide.

Interaction of Ca2+ and Gd3+ ions with Ca(2+)-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) was analyzed. Binding of Ca2+ to the transport site caused an enhancement of intrinsic fluorescence of SR-ATPase. Gd3+ also induced fluorescence enhancement. However, the effects of Ca2+ and Gd3+ were additive rather than competitive, indicating that the Gd(3+)-binding site responsible for this enhancement is distinct from the Ca(2+)-transport site. Gd3+ ions at concentrations higher than 10 microM caused a marked fluorescence quenching, indicating an additional interaction at low-affinity binding sites. Interaction of Ca2+ with the transport site led to a quenching of fluorescence of N-(1-anilinonaphthyl-4)maleimide (ANM) covalently attached at SHN [as defined in Yasuoka-Yabe, K. & Kawakita, M. (1983) J. Biochem. 94, 665-675]. In this case the effects of Ca2+ and Gd3+ were mutually exclusive, indicating that Ca2+ and Gd3+ were competing for the same binding site (i.e. the transport site) to affect ANM fluorescence. Competition between Ca2+ and Gd3+ for the Ca(2+)-transport site was also demonstrated by direct measurement of Ca(2+)-binding using nitrocellulose membrane filters. Affinity of Gd3+ for the Ca(2+)-transport site was a little lower than that of Ca2+. Based on these results it was concluded that Gd3+ has at least three kinds of binding sites on SR-ATPase, namely the Ca(2+)-transport site, the Gd(3+)-specific high-affinity site, and a number of low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aminopolycarboxylates of rare earths. 13. The apparent molal volumes of the lanthanide(III), cobalt(III) and chromium(III) ethylenediamine tetraacetate complexes

The apparent molal volumes of the complexes KLnedta (Ln = La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Er and Yb), KCoedta and KCredta were determined from density measurements. The apparent molal volumes of the complexes KLnedta show a non-monotonous change with increasing atomic number; there is a significant increase in the values between Nd and Gd. To explain the observed trend, a structural change is postulated to take place gradually on progressing from Gd towards La. With increasing ionic size, there is an increase in the metal-ligand bond distances and in the mobility of the functional groups, resulting in a jump in the number of coordinated water molecules by one between Gd and Nd. There is a further increase in the hydration of the central ions, connected with the gradual de-coordination of a carboxylate group. As a result of the gradual structural change there is one free carboxylate group on average in Laedta^? and about four water molecules are coordinated in the inner sphere, while in the complexes of the elements heavier than Gd the ligand is hexadentate and only two water molecules are in the inner sphere. The apparent molal volumes of KCoedta and KCredta are practically equal and are higher than those of the lanthanide complexes; this has been explained by the stronger hydration and the more significant water-ordering effect of the Ln³⁺ ions coordinated in the complexes Lnedta^?.The postulated structural changes correlate with the trend of the protonation constant values, as well as with the results of ¹H NMR studies on the complexes Lnedta^?.     

Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315.

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment...