Oxidative Nucleobase Modifications Leading to Strand Scission

Radiative decay engineering: biophysical and biomedical applications.

Determining the mechanism by which proteins attain their native structure is an important but difficult problem in basic biology. The study of protein folding is difficult because it involves the identification and characterization of folding intermediates that are only very transiently present. Disulfide bond formation is thermodynamically linked to protein folding. The availability of thiol trapping reagents and the relatively slow kinetics of disulfide bond formation have facilitated the isolation, purification, and characterization of disulfide-linked folding intermediates. As a result, the folding pathways of several disulfide-rich proteins are among the best known of any protein. This review discusses disulfide bond formation and its relationship to protein folding in vitro and in vivo.

Disulfide-Linked Protein Folding Pathways

Solute fluorescence quenching reactions were first applied to biochemical problems in the late 1960s and early 1970s, 7) and since that time they have been a very valuable research tool for studies with proteins, membranes, and other macromolecular assemblies. Quenching reactions are easy to perform, require only a small sample, usually are nondestructive, and can be applied to almost any system that has an intrinsic or extrinsic fluorescence probe. The most important characteristic, however, is the value of the information that these reactions can provide. Solute quenching reactions, using quenchers such as molecular oxygen, acrylamide, or iodide ion, provide information about the location of fluorescent groups in a macromolecular structure. A fluorophore that is located on the surface of a larger structure will be relatively accessible to a solute quencher that is dissolved in the aqueous phase. A fluorophore that is removed from the surface of a structure will be quenched to a lesser degree by the quencher. Thus, the quenching reaction can be used to probe topographical features of a macromolecular assembly and to sense any structural changes that may be caused by varying conditions or the addition of reagents. In addition, quenching reactions can, in some situations, provide information about conformational fluctuations. In Sections 2.3 and 2.4 I will discuss several examples of the use of solute quenchers in studies with proteins, membranes, and nucleic acids. Solute fluorescence quenching reactions can also be used to selectively alter the fluorescence properties of a sample in order to resolve contributions or aid in the measurement of data. To elaborate on this point, consider the different characteristics of fluorescence: the quantum yield, excitation and

Fluorescence Quenching: Theory and Applications

https://ddd.uab.cat/pub/artpub/2006/67768/PREI2008_trebiosci.pdf

Folding of small disulfide-rich proteins: clarifying the puzzle.

A mechanism was proposed several years ago for the regeneration of native ribonuclease A (EC 3.1.27.5) from the fully reduced form by a mixture of oxidized and reduced glutathiones. Several folding pathways, depending on the solution conditions, were deduced. It is shown here that recent criticisms of those results are due to a misinterpretation of the analysis of our data. A more detailed description of our method of analysis of our previous kinetic and energetic data is presented in order to clarify possible misconceptions.

Toward an understanding of the folding of ribonuclease A

On the basis of two experimental observations, it is established that the refolding mechanism of ribonuclease A (RNase A) is independent of the nature of the denaturant used [urea or guanidine hydrochloride (Gdn.HCl)]. First, by use of a double-jump technique, it is demonstrated that a similar nativelike intermediate exists on the major slow-folding pathway of both urea- and Gdn.HCl-denatured RNase A. Second, from the temperature dependence of the slow-refolding kinetics, it is shown that the activation parameters (both enthalpy and entropy) of the rate-limiting steps, as monitored by tyrosine absorbance and fluorescence, are identical for the refolding of urea- and Gdn.HCl- denatured RNase A. A refolding scheme involving one intermediate on each of the two slow-folding pathways is proposed by adopting the notion that RNase A refolds through a sequential mechanism. However, these two intermediates are formed from their respective unfolded forms (USII and USI) through two different processes of distinct physical origin. The intermediate IN, which is formed from the major slow-folding species USII through a conformational folding step, already possesses many properties of the native protein. In contrast, the intermediate (designated as I') on the minor slow-folding pathway is formed from USI by the isomerization of a proline residue (possibly Pro93) and is still conformationally unfolded. It is shown that such a refolding scheme can account for the known kinetic features of both major and minor slow-refolding pathways of RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and mechanism of the refolding of denatured ribonuclease A

Tyrosyl fluorescence quenching by oxidized dithiothreitol (DTTo) in N-acetyl-L-tyrosine N'-methylamide, and native bovine pancreatic ribonuclease A and its reduced, S-methylated form, in aqueous solution is studied at pH 3.0. From the temperature dependence of the fluorescence quenching, it is demonstrated that the mechanism of the quenching process is probably static (formation of a complex), and not dynamic (collisional), in origin. Although other quenching mechanisms cannot be ruled out, our proposition that the quenching of tyrosyl fluorescence in these molecules is due to the formation of a complex between the tyrosyl moieties and DTTo is consistent with previously reported evidence indicating a strong tendency for aromatics to complex with various disulfide-containing compounds. The strength of binding is approximately the same for these three tyrosine-containing compounds, indicating that the microenvironments of their tyrosyl residues may be similar. With 1 M as the reference standard state, the following average thermodynamic parameters are established for the complexation (at 298 K): delta G0 = -3.32 kcal/mol, delta H0 = -1.1 kcal/mol, and delta S0 = 7.4 eu. The large positive value of delta S0 suggests that hydrophobic interactions may play an important role in the stabilization of such tyrosyl-disulfide complexes; the negative value of delta H0 suggests that polar interactions may also contribute to the formation of these complexes. Some possible implications with regard to protein-folding studies are discussed.

Thermodynamics of the quenching of tyrosyl fluorescence by dithiothreitol.

The intrinsic component of the standard free energy change for the formation of a disulfide bond in a protein molecule is compared to that for an analogous chemical reaction. The former reaction, which represents theintramolecular formation of a disulfide bond in a protein molecule from a cysteine group containing a mixed disulfide bond with glutathione, and a free cysteine residue, is a unimolecular reaction. In contrast, its chemical analogue is a bimolecular reaction, and corresponds to theintermolecular disulfide interchange between a mixed disulfide-bonded compound between a cysteine residue and glutathione, and a free cysteine molecule. The difference in the intrinsic free energy of the above two reactions is estimated by two different approaches. First, a theoretical estimate of the magnitude of the difference in free energy of the two reactions (for a standard state of 1 M) is obtained using a gas-phase statistical thermodynamic approach, which indicates that the intramolecular reaction is energetically favored over its intermolecular counterpart by as much as 15.6 kcal/mole. For comparison, an experimentally derived value is also obtained, using experimental data from a study by Konishi et al. of the regeneration of the protein ribonuclease A (RNase A) from its reduced form by reduced and oxidized glutathiones. The intrinsic component of the free energy change of the intramolecular reaction, as it occurs in the protein molecule, is obtained from such experimental data by accounting explicitly for the free energy change (assumed to be solely an entropy change) pertaining to the conformational changes (ring closure) that the protein molecule undergoes in the course of the reaction. On the basis of the value derived from such an experimental approach, the intramolecular reaction is also energetically more favorable as compared to its intermolecular analogue, but only by a difference of 2.3 kcal/mole (for a standard state of 1 M). The large apparent discrepancy between the two values estimated from the theoretical and experimental approaches is rationalized by the postulation of several additional factors not inherent in the gas-phase theoretical estimate, such as dehydration and intramolecular hydrogen-bonding effects, which can largely compensate for the otherwise favorable energetics of the intramolecular reaction.

Comparison of intramolecular and intermolecular reactions in protein folding

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Toward an understanding of the folding of ribonuclease A

Kinetics and mechanism of the refolding of denatured ribonuclease A

Thermodynamics of the quenching of tyrosyl fluorescence by dithiothreitol.

Comparison of intramolecular and intermolecular reactions in protein folding