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Principles Of Fluorescence Spectroscopy

Initiation of translation in prokaryotes and eukaryotes.

Radiative decay engineering: biophysical and biomedical applications.

This article presents evidence for the existence of a specific linear relationship between the entropy change and the enthalpy change in a variety of processes of small solutes in water solution. The processes include solvation of ions and nonelectrolytes, hydrolysis, oxidation–reduction, ionization of weak electrolytes, and quenching of indole fluorescence among others. The values of the proportionality constant, called the compensation temperature, lie in a relatively narrow range, from about 250 to 315 °K, for all these processes. Such behavior can be a consequence of experimental errors but for a number of the processes the precision of the data is sufficient to show that the enthalpy–entropy compensation pattern is real. It is tentatively concluded that the pattern is real, very common and a consequence of the properties of liquid water as a solvent regardless of the solutes and the solute processes studied. As such the phenomenon requires that theoretical treatments of solute processes in water be expanded by inclusion of a specific treatment of the characteristic of water responsible for compensation behavior. The possible bases of the effect are proposed to be temperature-independent heat-capacity changes and/or shifts in concentrations of the two phenomenologically significant species of water. The relationship of these alternatives to the two-state process of water suggested by spectroscopic and relaxation studies is examined. The existence of a similar and probably identical relationship between enthalpy and entropy change in a variety of protein reactions suggests that liquid water plays a direct role in many protein processes and may be a common participant in the physiological function of proteins. It is proposed that the linear enthalpy–entropy relationship be used as a diagnostic test for the participation of water in protein processes. On this basis the catalytic processes of chymotrypsin and acetylcholinesterase are dominated by the properties of bulk water. The binding of oxygen by hemoglobin may fall in the same category. Similarities and differences in the behavior of small-solute and protein processes are examined to show how they may be related. No positive conclusions are established, but it is possible that protein processes are coupled to water via expansions and contractions of the protein and that in general the special pattern of enthalpy–entropy compensation is a consequent of the properties of water which require that expansions and contractions of solutes effect changes in the free volume of the nearby liquid water. It is shown that proteins can be expected to respond to changes in nearby water and interfacial free energy by expansions and contractions. Such responses may explain a variety of currently unexplained characteristics of protein solutions. More generally, the enthalpy–entropy compensation pattern appears to be the thermodynamic manifestation of “structure making” and “structure breaking,” operationally defined terms much used in discussions of water solutions. If so, the compensation pattern is ubiquitous and requires re-examination of a large body of molecular interpretations derived from quantitative studies of processes in water. Theories of processes in water may have to be expanded to accommodate this aspect of water behavior.

Enthalpy-entropy compensation phenomena in water solutions of proteins and small molecules: a ubiquitous property of water.

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

https://www.pnas.org/content/pnas/96/11/6171.full.pdf

Thermodynamic Basis of the Enhanced Specificity of Structured DNA Probes

A single 16-base oligodeoxyribonucleotide was labeled at the 3'-end with fluorescein and at the 5'-end with x-rhodamine (R*oligo*F); the chromophores served as a donor/acceptor pair, respectively, for Forster resonance energy transfer. We exploited the striking differences in the steady-state emission spectra of the R*oligo*F as a single strand and in a duplex structure to signal hybridization in solution and to determine the kinetics of duplex formation as the probe bound to its oligomer complement and to its target sequence in M13mp18(+) phage DNA. The binding followed second-order kinetics; in 0.18 M NaCl (pH 8) with 25% formamide, the rate constant for binding to the oligomer complement was 5.7 x 10(5) M-1 s-1, and that to M13mp18(+) was 5.7 x 10(4) M-1 s-1. The source of the 10-fold decrease in the rate of binding to M13mp18(+) was examined to differentiate between multiple nonproductive nucleation and rapid fluctuations in the structure around the target site. From simulations based on each model combined with associated experimental results, we concluded that the slower binding was due to rapid structural fluctuations around the target site, with an effective target concentration 0.1 of that of the total. Comparisons of total fluorescein emission derived from both steady-state and lifetime measurements suggest that the 5'-x-rhodamine induces a conformational change that affects the interaction at the 3'-end between the fluorescein and the polymer. The effects of salt on the fluorescence were complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic Studies by Fluorescence Resonance Energy Transfer Employing a Double-Labeled Oligonucleotide: Hybridization to the Oligonucleotide Complement and to Single-Stranded DNA

http://www.jbc.org/content/244/17/4668.full.pdf

The Reaction of Methemoglobin with Some Ligands

The binding and bending of tetramethylrhodamine-5'-(GGGCTATAAAAGGG) duplex-3'-fluorescein by native Saccharomyces cerevisiae TATA binding protein (TBP) have been investigated using fluorescence resonance energy transfer. Probability distributions derived from fluorescein emission lifetime measurements show a decrease in the mean 3'-fluorescein-5'- rhodamine distance from 56.5 to 46.8 A upon binding of the oligomer to TBP, consistent with the DNA bend observed by X-ray crystallography. The kinetics, monitored in real time using stopped flow fluorimetry, demonstrate simultaneous binding and bending of a TATA box by TBP with a single second-order rate constant of (2.4 +/- 0.3) x 10(6) M-1 s-1 at 30 degrees C.

Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements.

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1003&context=chemistryparkhurst

Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex.

A 16-mer deoxyribonucleotide was labeled at the 5'-end with x-rhodamine and at the 3'-end with fluorescein. Forster resonance energy transfer was used to determine the distribution, P(R), of donor-acceptor distances for the oligomer in three duplex structures (hybridized to complementary strands having 10, 16, and 24 bases) and as a single strand; measurements were made in 0.18 M NaCl and 1 M KCl solutions. These distributions were derived from lifetime measurements made in the frequency domain using a multiharmonic frequency phase fluorometer. The fluorescein fluorescence decay for each duplex structure was fit very well with P(R) modeled as a shifted Gaussian. On the basis of the mean donor-acceptor distance, these structures for the 16-mer and the 24-mer were consistent with that of B-DNA. For the 16-mer duplex in 0.18 M NaCl, the distribution was centered at 68.4 A with sigma = 6.4 A. For the 10-mer duplex, the mean donor-acceptor distance, 61.8 A, is much larger than that for a structure with the fluorophores extended perpendicular to the helix axis. For the single-strand data, various high-quality fits yielded physically unreasonable distributions or could not accurately account for the acceptor response. Considered analyses suggested that the single-strand distribution was best represented by a shifted Gaussian, with R = 51.5 A and sigma = 10.0 A in 0.18 M NaCl. In all cases, sigma increased and R decreased in 1 M KCl relative to their values in 0.18 M NaCl, consistent with the increased flexibility of the polymer.

Lawrence J. Parkhurst

Papers

Kinetic Studies by Fluorescence Resonance Energy Transfer Employing a Double-Labeled Oligonucleotide: Hybridization to the Oligonucleotide Complement and to Single-Stranded DNA

The Reaction of Methemoglobin with Some Ligands

Simultaneous binding and bending of promoter DNA by the TATA binding protein: real time kinetic measurements.

Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex.

Donor- acceptor distance distributions in a double-labeled fluorescent oligonucleotide both as a single strand and in duplexes