Nanosecond T-Jump Experiment in Poly(glutamic acid): A Circular Dichroism Study

Time-resolved near UV circular dichroism and absorption studies of carbonmonoxymyoglobin photolysis intermediates

Optical activity is the result of chiral molecules interacting differently with left versus right circularly polarized light. Because of this intrinsic link to molecular structure, the determination of optical activity through circular dichroism (CD) spectroscopy has long served as a routine method for obtaining structural information about chemical and biological systems in condensed phases. A recent development is time-resolved CD spectroscopy, which can in principle map the structural changes associated with biomolecular function and thus lead to mechanistic insights into fundamental biological processes. But implementing time-resolved CD measurements is experimentally challenging because CD is a notoriously weak effect (a factor of 10(-4)-10(-6) smaller than absorption). In fact, this problem has so far prevented time-resolved vibrational CD experiments. Here we show that vibrational CD spectroscopy with femtosecond time resolution can be realized when using heterodyned spectral interferometry to detect the phase and amplitude of the infrared optical activity free-induction-decay field in time (much like in a pulsed NMR experiment). We show that we can detect extremely weak signals in the presence of large achiral background contributions, by simultaneously measuring with a femtosecond laser pulse the vibrational CD and optical rotatory dispersion spectra of dissolved chiral limonene molecules. We have so far only targeted molecules in equilibrium, but it would be straightforward to extend the method for the observation of ultrafast structural changes such as those occurring during protein folding or asymmetric chemical reactions. That is, we should now be in a position to produce 'molecular motion pictures' of fundamental molecular processes from a chiral perspective.

The early dynamics of reduced cytochrome c (cytc) folding initiated by a photoinduced electron-transfer reaction are studied using time-resolved circular dichroism spectroscopy. At 3.5 M GdnHCl oxidized cytc is partly unfolded, whereas the reduced form is folded. Thus, under these conditions, rapid electron injection into unfolded, oxidized cytc triggers folding. The kinetics of secondary structure formation in reduced cytc occurs in two major phases. The earliest detection of reduction is accompanied by the appearance of 20% of the secondary structure within 5 μs. From time-resolved absorption and circular dichroism studies this rapid folding is ascribed to a subpopulation of unfolded protein molecules that have a structural tendency to form a native His18-Fe(II)-Met80 ligation. Thereafter, almost no change in the secondary structure is observed until the CD signal starts to decrease in magnitude between 16 μs and 1 ms. This “unfolding” phase (τ ≈ 180 μs) is followed by a small increase in the magnitude ...

Time-resolved circular dichroism (TR-CD) is a powerful tool for probing conformational dynamics of biomolecules over large time scales that are crucial for establishing their structure-function relationship. However, such experiments, notably in the femtosecond regime, remain challenging due to their extremely weak signals, prone to polarization artifacts. By using binol and two bridged derivatives (PL1 and PL2) as chiral prototypes, we present here the first comprehensive study of this type in the middle UV, combining femtosecond TR-CD and quantum mechanical calculations (TD-DFT). We show that excitation of the three compounds induces large variations of their transient CD signals, in sharp contrast to those of their achiral transient absorption. We demonstrate that these variations arise from both the alteration of the electronic distribution and the dihedral angle in the excited state. These results highlight the great sensitivity of TR-CD detection to signals hardly accessible to achiral transient absorption.

Absorption spectra and circular dichroism (CD) of aqueous solutions of a water-soluble porphyrin derivative, porphine-meso-tetra(4-N-methylpyridinium) tosylate (TMpyP tosylate), have been measured in the presence of poly(l-glutamic acid) or poly(S-carboxymethyl-l-cysteine) at different pH. At neutral and alkaline pH where poly(l-glutamic acid) is randomly coiled, essentially no CD is induced in the Soret region over a wide range of [P]/[D] ratios. At acid pH where poly(l-glutamic acid) is helical, a positive CD band at 415 nm and a stronger negative CD band at 442 nm are induced if [P]/[D] is high, but the induced CD is considerably weak as [P]/[D] is lowered, owing to partial disruption of the helical conformation by densely bound TMpyP ions, and it substantially vanishes at [P]/[D] as low as 1. While random coil poly(S-carboxymethyl-l-cysteine) does not induce CD in the Soret region of TMpyP, β-form poly(S-carboxymethyl-l-cysteine) induces a weak, positive CD band at 510 nm, accompanied by an extensive weakening of the absorption bands.

Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio

UV-Visible Absorption and Circular Dichroism Spectroscopy, Inorganic Chemistry Applications

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ-HIgG complex. Fluorescence spectroscopy, ultraviolet-visible (UV-vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH(0) and entropy ΔS(0), had been calculated to be -19.12 kJ · mol(-1) and 20.22 J · mol(-1) · K(-1), respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ - HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three-dimensional fluorescence spectroscopy, UV-vis absorption spectroscopy and CD spectroscopy.

Classification of CD and absorption spectra in the Soret band of H 2TMPyP bound to various synthetic polynucleotides

Specificity and affinity of phenosafranine protein adduct: Insights from biophysical aspects

Poly(l-arginine) (PARG) and poly(l-glutamic acid) (PLGA) homopolypeptides were custom synthesized by precision N-carboxyanhydride ring-opening polymerization methods with two molar masses, matched for pairs of cationic and anionic polypeptides (degrees of polymerization n = 100 and n = 500). The conformations of the homopolypeptides were probed using circular dichroism (CD) and FTIR spectroscopy which revealed the presence of mainly polyproline II (PPII) conformation. Small-angle X-ray scattering (SAXS) showed concentration-dependent polyelectrolyte peaks and form factor with high q scaling due to the excluded volume behaviour of the wormlike chains. We then examined polyelectrolyte complexation in mixtures of pairs of PARG and PLGA polypeptides with matched molar masses. Precipitation was generally observed and the structures of precipitates, supernatant and resuspended precipitates were investigated using CD, SAXS and cryo-TEM. These revealed that, contrary to prior suggestions in the literature, the precipitates contain mostly polypeptides in a PPII-like conformation, and there is only a minimal β-sheet content (which is enhanced upon drying the sample during preparation for certain measurements). The precipitates have a fractal-like structure as revealed by cryo-TEM and SAXS. Our findings on the structure of polypeptide complex precipitates contribute to the understanding of phase separation of polyelectrolyte complexes and coacervation and may shed light on the formation of inter-cellular bodies of proteins and peptides such as Lewy and other inclusion bodies.

Different effects of subunit association upon absorption and circular dichroism spectra of methemoglobin

Spectroscopic probing of ultrafast chiral molecular dynamics gives deep insight into the mechanism of biomolecular reactions, since the three-dimensional molecular structure plays crucial roles in the selectivity and efficiency of the reactions. Here, we demonstrate a practical method for tracking the change of the absolute configuration of photoexcited biomolecules by combining ultrafast and broadband time-resolved circular dichroism (TRCD) spectroscopy with exciton coupling theory. A proof-of-principle experiment on bilirubin-human serum albumin (BR-HSA) complexes reveals that the excited-state CD spectra show sign inversion with a time constant of ∼5 ps. Instead of a complete chirality inversion across a high energy barrier, we attribute this sign reversal to a structural change from the stable ridge-tile conformation to a stretched conformation based on exciton coupling theory. These results show that the present method is a useful tool for probing excited-state molecular dynamics accompanying structural changes on a real-time basis.

Circular dichroism and absorption spectra have been measured on solutions of acridine orange and poly(L‐glutamic acid) mixed at two molar ratios of carboxyl group‐to‐dye, P/D 25 and 0.8, and at different pH's. Characteristic circular dichroism is induced at the absorption bands of acridine orange when the P/D is 25 and the solution is acidic. Another type of circular dichroism is manifest at neutral and alkaline pH when P/D is 0.8. For the induction of the former type of circular dichroism, a helical array of acridine‐orange dimers bound to the α‐helix is postulated, in which the dye molecular planes are almost perpendicular to the helical axis. Assuming the helical geometry and optical parameters, combined with the observed magnitude of transition electric moment, the rotatory strength of the complex is calculated to the zeroth order approximation, and the observed circular dichroism spectra have been reproduced.

The static infrared absorption (IR) and circular dichroism (CD) spectra of the temperature-dependent conformational states of apomyoglobin in potassium phosphate and sodium acetate buffer solution at pH 5.3 in D 2 O are reported. The circular dichroism ellipticity at 222 nm reveals a loss of helical structure for both heat- and cold-denatured states. Cold denaturation leads to a molten globule state with an associated partial loss of helical structure identified at 1646 cm −1 . Heat denaturation also exhibits an absorption loss at 1646 cm −1 but leads to the appearance of aggregation at 1680 and 1619 cm −1 . There is semi-quantitative agreement of the ultraviolet circular dichroism and infrared estimates of protein helicity. The denaturation spectra give evidence that there are specific associations between acetate ions and the protein, but not between phosphate ions and the protein.