Enoch W. Small

Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump.

Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of -82 +/- 3 ml/mol, an enthalpy change of 8 +/- 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 +/- 0.03) x 10(10) M(-1) s(-1). Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0. 60 +/- 0.07) x 10(6) s(-1). The second protonation reaction at pH 7. 0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 +/- 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 +/- 0.02) x 10(11) M(-1) s(-1), and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 +/- 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 +/- 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation.

Kinetics of Local Helix Formation in Poly-L-Glutamic Acid Studied by Time-Resolved Photoacoustics: Neutralization Reactions of Carboxylates in Aqueous Solutions and Their Relevance to the Problem of Protein Folding

Photoactivatable caged protons have been used to trigger proton transfer reactions in aqueous solutions of acetate, glutamate, and poly-L-glutamic acid, and the volumetric and enthalpic changes have been detected and characterized by means of time-resolved photoacoustics. Neutralization of carboxylates in aqueous solutions invariably results in an expansion of the solution due to the disappearance of two charges and is accompanied by little enthalpic change. The reactions occur with thermally activated, apparent bimolecular rates on the order of 10(10) M(-1)s(-1). In the case of aqueous solutions of poly-L-glutamic acid at pH around the pK(a) of the coil-to-helix transition, diffusional binding of a proton by carboxylates is followed by a sequential reaction with rate 1.06 (+/- 0.05) x 10(7)s(-1). This step is not thermally activated in the temperature range we have investigated and is likely related to local formation of hydrogen bonds near the protonation site. This structural event may constitute a rate-limiting step in helix propagation.

Histone hyperacetylation. Its effects on nucleosome core particle transitions

Effects of histone hyperacetylation on transitions of HeLa cell nucleosome core particles were studied. The transitions examined were induced by low salt concentrations, pH, temperature, and nondissociating high salt. Effects of salt dissociation were also examined. The low-salt transition was found to shift to higher ionic strength by approximately three fold for hyperacetylated particles, a change which may be due simply to the increased overall negative charge on the particles caused by acetylation of lysine residues. Some differences were also seen in the way in which core particles refold after exposure to very low salt (which induces a nonreversible change in the particles). Otherwise no significant effects of hyperacetylation were observed.

On moment index displacement

Moment index displacement can determine the amount of light scatter and zero point displacement in fluorescence time decay data. Two different types of light scatter are considered and shown to have significantly different effects on the analysis of the data. The amplitude correction factor determined by Eisenfeld and Mishelevich is experimentally verified.

FF deconvolution of fluorescence decay data

An approach for the deconvolution of multiexponential fluorescence decay data in which a single exponential decay is used in place of the usual excitation profile is described. For analysis by the method of moments, the resulting decay lifetimes are identical to those in the multiexponential decay, while the pre-exponential factors are a simple function of the true values and the parameters of the single exponential decay. This approach, which we call the F/F deconvolution method, is capable of eliminating the errors in decay analyses which arise from the wavelength dependence of the instrument response function.

Exponential depression as a test of estimated decay parameters

A new test for judging the goodness of estimated decay parameters is presented. The test is based on the fact that a convolution is invariant under exponential depression. In the absence of significant error the estimated parameters will then remain constant as the degree of depression is varied over a finite range. In the presence of error, the parameters will vary. Up to now, no test has existed to see if moment index displacement corrects errors to a satisfactory extent in any given analysis. It has always been necessary to have some a priori knowledge of the type of error that limited the analysis. The test presented here removes that requirement. In addition, it is shown that the test performs better than a visual inspection of residual and autocorrelation plots in judging analyses when decays are closely spaced, even in the absence of nonrandom errors. The test is useful in accepting or rejecting analyses, with or without automatic error correction, in helping to discriminate between different model...

Construction and tuning of a monophoton decay fluorometer with high‐ resolution capabilities

A fluorescence decay instrument, assembled largely from commercially available components, is described. This system incorporates a tunable picosecond laser system as an excitation source, energy windowing to minimize artifacts due to pulse pile up and sample alternation to compensate for long‐term instrumental drift. The instrument was tested for its ability to resolve multiple exponential decays using methods of moments analysis. By examining the flatness of λ‐invariance plots and the accuracy of recovered decay parameters, proper tuning of electronic components and definition of a range of reasonable counting rates was possible. Errors of significance to the resolution of closely spaced decays are discussed. A number of examples are shown for resolutions that are generally regarded as difficult, including two, three, and four components.

Application of method of moments analysis to fluorescence decay lifetime distributions

In fluorescence decay work, distributions of exponential decay lifetimes are anticipated where complex systems are examined. We describe here methods of gaining information on such distributions using the method of moments analysis approach. The information obtained may be as simple as the average and deviation of the lifetime distribution, quantities which we show may be estimated directly from the results of a multiexponential analysis. An approximation to the actual distribution shape may also be obtained using a procedure we call the variable filter analysis (VFA) method without making any assumptions about the shape of the distribution. Tests of VFA using both simulated and experimental data are described. Limitations of this method and of distribution analysis methods in general are discussed. Results of analyses on experimental decays for ethidium intercalated in core particles and in free DNA are reported.

An experimental methodology for measuring volume changes in proton transfer reactions in aqueous solutions

A fast perturbation in proton concentration can be induced in aqueous solution using a pulsed ultraviolet laser and suitable photolabile compounds which, upon photoexcitation, irreversibly release protons. The volume change and the rate constant for the reaction of the photodetached protons with proton-accepting groups in solution can be monitored using time resolved photoacoustics. A typical proton concentration jump of 1 microM can be obtained with a 200-microJ laser pulse at 308 nm. Reaction dynamics from 20 ns to 5 micros can be easily followed. The methodology we establish represents a direct, time-resolved measurement of the reaction volume in proton transfer processes and an extension to the nanosecond-microsecond range of traditional relaxation techniques, such as stopped-flow. We report example applications to reactions involving simple molecules and polypeptides.

Resolution of closely spaced fluorescence decays—the luminescence background of the RCA 8850 photomultiplier and other sources of error

The RCA 8850 photomultiplier has a wavelength‐dependent luminescence background when used in the UV below 320 nm. We show that this luminescence gives rise to an additional decay in the deconvoluted fluorescence and, unless corrected for, seriously interferes with the resolution of closely spaced fluorescence decays. A much lower luminescence background is found for the RCA C31000M photomultiplier, which is identical to the 8850 except that it has a fused silica rather than a Pyrex window. Use of the C31000M improves the resolution of known mixtures, but the recovered lifetimes are still somewhat in error. We believe that this residual error is due to additional low‐amplitude components in the fluorescence which cannot be resolved. An approach is presented which minimizes the effects of these contaminant decays on an analysis. Using this method, the decays of mixed compounds with lifetimes of 8.8 and 12.8 ns, of 10.1 and 12.8 ns, and of 8.8 and 10.1 ns are accurately resolved.