Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate.

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model.

Perchlorate-induced conformational transition of Staphylococcal nuclease: evidence for an equilibrium unfolding intermediate.

The sodium perchlorate-induced conformational transition of Staphylococcal nuclease has been monitored by both circular dichroism (CD) and fluorescence spectroscopy. The perchlorate-induced transition is cooperative as observed by both spectroscopic signals. However, the protein loses only about one-third of its native far-UV CD signal at high perchlorate concentrations, indicating that a significant amount of secondary structure remains in the post-transition state.

Activation of horse liver alcohol dehydrogenase upon substitution of tryptophan 314 at the dimer interface.

Horse liver alcohol dehydrogenase contains two tryptophan residues per subunit, Trp-15 on the surface of the catalytic domain and Trp-314 buried in the interface between the subunits of the dimer. We studied the contributions of the tryptophans to fluorescence and catalytic dynamics by substituting Trp-314 with a leucine residue and making two compensatory mutations that were required to obtain a stable protein, leading to the triple mutant M303F-L308I-W314L enzyme. The substitutions increased by two- to sixfold the turnover numbers for ethanol oxidation, acetaldehyde reduction, and the dissociation constants of the coenzymes.

Phosphorescence and optically detected magnetic resonance characterization of the environments of tryptophan analogues in staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment.

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on the triplet states of the tryptophan analogues, 7-azatryptophan (7AW), 5-hydroxytryptophan (5HW), and 4-, 5-, and 6-fluorotryptophan (4FW, 5FW, 6FW), when incorporated at position 140 of wild-type Staphylococcal nuclease (7AW-nuclease, etc. ), positions 66 and 140 of its V66W mutant (7AW-V66W, etc.), and the deletion fragment of the latter, Delta 137-149 (7AW-V66W', etc.

Incorporation of tryptophan analogues into staphylococcal nuclease: stability toward thermal and guanidine-HCl induced unfolding.

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been biosynthetically incorporated into Staphylococcal nuclease, its V66W mutant, and the Delta 137-149 fragment of the latter mutant. The guanidine-HCl induced unfolding and thermal unfolding of these proteins were studied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three proteins have tryptophan residues at positions 140 (in wild type) and 66 (in the Delta 137-149 fragment of V66W) and at both positions (in V66W).

Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment: spectroscopic studies.

We have biosynthetically incorporated several tryptophan analogues into three forms of Staphylococcal nuclease to investigate the spectroscopic characteristics of these "intrinsic" probes and their effect on the structure of the proteins. The set of tryptophan analogues includes 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan. 5-Hydroxytryptophan and 7-azatryptophan have red-shifted absorbance spectra, and the latter has a red-shifted fluorescence, which is very sensitive to its environment (being heavily quenched in water).

The use of fluorescence methods to monitor unfolding transitions in proteins.

The advantages and some limitations of the use of fluorescence methods for the quantitative determination of the thermodynamics of protein unfolding transitions (i.e., induced by temperature or chemical denaturant) are discussed.

Studies of the unfolding of an unstable mutant of staphylococcal nuclease: evidence for low temperature unfolding and compactness of the high temperature unfolded state.

Fluorescence and circular dichroism data as a function of temperature were obtained to characterize the unfolding of nuclease A and two of its less stable mutants. These spectroscopic data were obtained with a modified instrument that enables the nearly simultaneous detection of both fluorescence and CD data on the same sample. A global analysis of these multiple datasets yielded an excellent fit of a model that includes a change in the heat capacity change, deltaC(p), between the unfolded and native states.

Characterization of the role of side-chain interactions in the binding of ligands to apo trp repressor: pH dependence studies.

The pH dependence of the association of apo trp repressor with the series of ligands, tryptophan, tryptamine, indole propionic acid (IPA), and trans-beta-indole acrylic acid (IAA), has been studied using fluorescence titrations and isothermal titration microcalorimetry (ITC). The purpose of such a comparison of ligands and the pH dependency studies is to reveal the role played by the side-chain functional groups in the energetics of the binding of the ligands to the protein. We find that, whereas the binding of tryptamine and IPA have essentially no pH dependence between pH 6 and 10, the binding of tryptophan and IAA depends on pH.

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability.

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency.

Thermodynamics of protein unfolding: questions pertinent to testing the validity of the two-state model.

We discuss a number of questions pertaining to the analysis of data to extract thermodynamic parameters for the reversible unfolding of proteins. Simulations are presented to illustrate problems in trying to test the validity of the two-state model, vis-a-vis a more complicated unfolding model. A conceptual and practical problem is how to consider the unfolded state and how to relate the observed signal to this state.

Global analysis of the acid-induced and urea-induced unfolding of staphylococcal nuclease and two of its variants.

We have studied the equilibrium unfolding staphylococcal nuclease and two of its variants, V66W and V66W', over two perturbation axes (acid-induced unfolding as a function of urea concentration and urea-induced unfolding as a function of pH). The transitions were monitored by simultaneous measurements of circular dichroism and fluorescence. With this multidimensional array of data (2 perturbation axes and 2 signals), we present a strategy of performing a global analysis, over as many as 12 individual data sets, to test various models for the unfolding process, to determine with greater confidence the pertinent thermodynamic parameters, and to characterize unfolding intermediates.

Time-resolved fluorescence studies of ribonuclease T1 in reversed micelles.

Time-resolved fluorescence intensity and anisotropy decay data were obtained for ribonuclease T1 entrapped in bis(2-ethylhexyl) sodium sulfosuccinate/heptane reverse micelles, as a function of the size of the inner water pool at neutral pH. Data have been presented previously to show that this protein retains its native structure and undergoes reversible thermal unfolding in these reverse micelles (Shastry and Eftink,Biochemistry 36, in press). The fluorescence decay of entrapped protein is similar to that for the protein in buffer.

Thermodynamics of the unfolding and spectroscopic properties of the V66W mutant of Staphylococcal nuclease and its 1-136 fragment.

Spectroscopic studies have been performed to characterize the solution structure of the V66W mutant of Staphylococcal nuclease and the corresponding 1-136 fragment, referred to as V66W'. Whereas wild-type nuclease has a single tryptophan residue at position 140, the V66W mutant has a second tryptophan residue at position 66, which is the only such residue in V66W'. Steady-state and time-resolved fluorescence studies show Trp-66 in V66W' to have a blue emission, a relatively large fluorescence quantum yield, a long lifetime, a significant degree of protection from solute quenchers, and to depolarize with a relatively long rotational correlation time.

Reversible thermal unfolding of ribonuclease T1 in reverse micelles.

The reverse micellar system formed by the negatively charged surfactant AOT and the organic solvent isooctane is used to solubilize the protein RNase T1. The physicochemical properties of the entrapped protein have been studied using intrinsic tryptophan fluorescence and far-and near-UV CD. These studies indicate a similar structure for the protein in reverse micelles and in pH 7.

Characterization of the tryptophan binding site of Escherichia coli tryptophan holorepressor by phosphorescence and optical detection of magnetic resonance of a tryptophan-free mutant.

The L-tryptophan binding site of the Escherichia coli tryptophan holorepressor (trpR) is characterized by low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Measurements are made on a tryptophan-free mutant of trpR, W19/99F, in which both intrinsic tryptophan residues of apo-trpR have been replaced with phenylalanine. Thus, essentially all of the phosphorescence that is observed from trpR originates from the bound L-tryptophan corepressor.

Modified spectrophotometer for multi-dimensional circular dichroism/fluorescence data acquisition in titration experiments: application to the pH and guanidine-HCI induced unfolding of apomyoglobin.

In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements.

The unfolding of trp aporepressor as a function of pH: evidence for an unfolding intermediate.

The urea-induced unfolding of trp aporepressor from Escherichia coli has been studied as a function of pH from 2.5 to 12.0 at 25 degrees C.

Oxygen fluorescence quenching studies with single tryptophan-containing proteins.

The work of Lakowicz and Weber [Biochemistry 12, 4161 (1973)] demonstrated that molecular oxygen is a powerful quencher of tryptophan fluorescence in proteins. Here we report studies of the oxygen quenching of several proteins that have a single, internal tryptophan residue. Among these are apoazurin (Pseudomonas aeruginosa), asparaginase (Escherichia coli), ribonuclease T1 (Aspergillus oryzae), and cod parvalbumin.

Evidence for dual La and L b emission in 5-methylindole.

The steady-state fluorescence anisotropy of 5-methylindole is shown to depend on both excitation and emission wavelengths, at room temperature in glycerol. A simulation is presented that shows that this emission wavelength dependence of the anisotropy can be explained in terms of dual emission from both the La and the Lb transition moments of the indole ring. For such dual emission to occur, the lowest excited-state energy level of both of these oscillators must be very similar.

Thermodynamic studies of the interaction of trp aporepressor with tryptophan analogs.

The association of L-tryptophan and some of its analogs, including three conformationally restricted analogs, with trp aporepressor (apo trpR) was studied by isothermal titration microcalorimetry. Contributions of the functional groups of a ligand to the free energy change, delta G degrees', and enthalpy change, delta H degree', of the interaction were evaluated on a molecular basis. Analogs without the alpha-amino group (i.