Stopped-flow Circular Dichroism Spectroscopy Research Articles

Kinetics of Zn-DNA binding structural changes measured through stopped-flow circular dichroism.

Divalent cations are known to cause measurable changes in the circular dichroism signals measuring DNA structure. Comparing transition metal divalent ions and alkaline divalent ions shows different structures as a function of concentration and ion identity. Here, we measured the time-dependent nature of this divalent ion binding and corresponding structural changes in the DNA structure using stopped-flow circular dichroism spectroscopy (CD). We found measurable differences between structural changes measured during zinc and magnesium ion binding to the DNA. Combined with CD spectral studies, these experiments give insight into the dynamics and kinetics underlying these processes.

Impact of linker histone in the formation of ambochlorin-calf thymus DNA complex: Multi-spectroscopic, stopped-flow, and molecular modeling approaches.

Objective(s):This study aimed to evaluate the role of the linker histone (H1) in the binding interaction between ambochlorin (Amb), and calf thymus DNA (ctDNA) as binary and ternary systems. Materials and Methods:The project was accomplished through the means of absorbance, fluorescence, stopped-flow circular dichroism spectroscopy, viscosity, thermal melting, and molecular modeling techniques.Results:Spectroscopic analysis revealed that although Amb was strongly bound to both ctDNA and ctDNA-H1, it showed a greater tendency to ctDNA in the presence of the linker histone. The obtained thermodynamic parameters revealed that both Amb-ctDNA and Amb-ctDNA-H1 interactions were spontaneous, endothermic, and entropy-favored, and hydrophobic interactions played the main role in the formation and stabilization of complexes. Analysis of the stopped-flow circular dichroism results revealed that the binding process of Amb-ctDNA and Amb-ctDNA-H1 required a time of more than 150 milliseconds to complete. Moreover, Amb-ctDNA complex formation was marginally decelerated in the presence of the linker histone. The docking results suggested that the presence of the linker histone may alter the binding sites of Amb from ctDNA minor grooves to major grooves. Conclusion:All quenching processes were governed by a dynamic mechanism. Additionally, Amb did not stabilize or induce considerable conformational changes in ctDNA and ctDNA-H1 complex upon binding. In silico molecular docking results confirmed that Amb was bound to the double-helical ctDNA and ctDNA-H1 via ctDNA grooves. In summary, some binding properties of the interactions between Amb and ctDNA change in the presence of the linker histone.

Fast Folding of the HIV-1 and SIV gp41 Six-helix Bundles

Human (HIV-1) and simian (SIV) immunodeficiency virus fusion with the host cell is promoted by the receptor-triggered refolding of the gp41 envelope protein into a stable trimer-of-hairpins structure that brings viral and cellular membranes into close proximity. The core of this hairpin structure is a six-helix bundle in which an inner homotrimeric coiled coil is buttressed by three antiparallel outer HR2 helices. We have used stopped-flow circular dichroism spectroscopy to characterize the unfolding and refolding kinetics of the six-helix bundle using the HIV-1 and SIV N34(L6)C28 polypeptides. In each case, the time-course of ellipticity changes in refolding experiments is well described by a simple two-state model involving the native trimer and the unfolded monomers. The unfolding free energy of the HIV-1 and SIV trimers and their urea dependence calculated from kinetic data are in very good agreement with data measured directly by isothermal unfolding experiments. Thus, formation of the gp41 six-helix bundle structure involves no detectable population of stable, partly folded intermediates. Folding of HIV-1 N34(L6)C28 is five orders of magnitudes faster than folding of its SIV counterpart in aqueous buffer: k on , HIV-1 =1.3×10 15 M −2 s −1 versus k on , SIV =1.1×10 10 M −2 s −1. The unfolding rates are similar: k off , HIV-1 =1.1×10 −5 s −1 versus k off , SIV= 5.7×10 −4 s −1. Kinetic m-values indicate that the transition state for folding of the HIV-1 protein is significantly more compact than the transition state of the SIV protein. Replacement of a single SIV threonine by isoleucine corresponding to position 573 in the HIV-1 sequence significantly stabilizes the protein and renders the folding rate close to that of the HIV-1 protein yet without making the transition state of the mutant as compact as that of the HIV-1 protein. Therefore, the overall reduction of surface exposure in the high-energy transition state seems not to account for different folding rates. While the available biological evidence suggests that refolding of the gp41 protein is slow, our study implies that structural elements outside the trimer-of-hairpins limit the rate of HIV-1 fusion kinetics.

The role of ligand binding in the kinetic folding mechanism of human p21(H-ras) protein.

p21(H-ras) plays a critical role in signal transduction pathways by cycling between an active, GTP/Mg2+ ternary complex and an inactive, GDP/Mg2+ complex. Urea-induced equilibrium unfolding studies [Zhang and Matthews (1998) Biochemistry 37, 14881-14890] have shown that GDP and Mg2+ play essential roles in stabilizing the protein. To probe the mechanism of folding and to examine the effects of these ligands on the kinetic folding reaction, unfolding and refolding experiments were performed at a variety of urea and ligand concentrations. A burst phase intermediate with substantial secondary structure and marginal stability was observed during refolding by stopped-flow circular dichroism spectroscopy. Three subsequent refolding phases were detected using a combination of absorbance, circular dichroism, and fluorescence spectroscopy. The fastest phase involves ligand binding and appears to directly form the fully folded enzyme. The intermediate and slow phases do not depend on either urea or ligand concentration under strongly refolding conditions and appear to reflect isomerization or rearrangement reactions. Double- jump experiments demonstrated that the intermediate and slow refolding phases both lead to the native conformation and correspond to parallel rather than sequential reactions. Unfolding is controlled by two phases that involve the release of the ligands when the ligands are in excess. At stoichiometric ligand concentrations, however, the rate-limiting steps in unfolding change from ligand release to isomerization or rearrangement reactions at high urea concentrations. Only the faster unfolding reaction is observed in the absence of Mg2+, suggesting that this reaction corresponds to the unfolding of the binary complex, p21(H-ras)*GDP. The slower unfolding reaction presumably corresponds to the unfolding of the ternary complex, p21(H-ras)*GDP. Mg2+. The kinetic data show that the refolding/unfolding of p21(H-ras) occurs through parallel channels that are strongly influenced by the binding/release of GDP and Mg2+ to/from a pair of native conformers.

Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy.

Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable biomolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidine-HCl induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mol dimer)-1 when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on both peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N2[symbol: see text]2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 3.3 x 10(-3) S-1, respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.

Kinetic study on the successive four-step reduction of Cyt c3.

A detailed kinetic study on the successive four-step reduction of cyt c3, which has four heme units in a single protein, III4 leads to III3II leads to III2II2 leads to III II3 leads to II4, was carried out by stopped-flow electronic spectroscopy (SF-UV) and stopped-flow circular dichroism spectroscopy (SF-CD). Based on the absorbance change vs. time and the ellipticity change vs. time at the characteristic CD, together with the electronic absorption of the enzyme, rate constants for the successive four electron transfer steps, k1-k4, were successfully estimated by computer simulation. The rate constants of the four steps (k1 = 19.8 s-1, k2 = 11.9 s-1, k3 = 8.9 s-1, and k4 = 1.6 s-1; 8.0 10(-4) M Na2S2O4) are quite different from the statistical values (4: 3: 2: 1), thus excluding the possibility of random reduction of hemes of equal reactivities. Instead, each heme has its own reactivity, probably dependent on its local environment. The value of k3 is somewhat higher than the statistical value, indicating the existence of an autoacceleration effect, although small. This autoacceleration is most probably due to a unique heme-heme and/or heme-environment interaction since unusual CD and electronic absorptions were observed at 350-400 nm at about the time corresponding.

Measurement of circular dichroism at millisecond time resolution: a stopped-flow circular dichroism system

A new system has been designed for measuring circular dichroism with resolution Delta A approximately 10-5, at approximately 2 ms time constant, and used for stopped-flow circular dichroism spectroscopy. Expressions are derived for signal-to-noise ratio and circular dichroism resolution including contributions from signal current and detector dark current shot noise, and thermal and amplifier noise. An experimental system has been constructed using a mercury arc, piezo-optical birefringence modulator (50 kHz) and silicon photodiode detector. Transient CD signals have been monitored for rapid reactions conducted in a stopped-flow mixing device with dead time less than 1 ms. Phase sensitive detection and analogue division are used for CD signal recovery. The resolution achieved is close to the theoretical prediction. Applications are indicated for the study of ligand-induced conformational changes in biological systems.