Second derivative fluorescence spectroscopy of tryptophan in proteins.

The second derivatives of N-acetyl-L-tryptophan amide (AcTrpNH(2)) fluorescence spectra were characterised in order to describe changes in the tryptophan environments of proteins. This tryptophan model compound was studied in several media with different degrees of hydrophobicity. The effect of tyrosines on the derivative spectra was also determined in situations in which both tyrosine and tryptophan were excited. An analysis of fluorescence second derivative spectra suggests that AcTrpNH(2) fluorescence emission is composed of two main bands. Increasing solvent polarity resulted in a red-shift by both bands and a relative increase in the emission efficiency of the shortest wavelength band. The applicability of fluorescence second derivative is shown through several examples. Turbidity observed in whole membrane extracts, for example, is eliminated by using second derivative spectra. Melittin, human and bovine serum albumins and the carboxypeptidase-PCI complex were studied as examples of the use of fluorescence second derivative spectroscopy to monitor changes in structural characteristics when these proteins were subjected to various transitions.

Rapid formation and high diffusibility of actin-cofilin cofilaments at low pH.

Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (DLM) as well as the fraction of polymerized actin, fLM, present in solution. In the range of cofilin-actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP-actin-cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin-actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin-cofilin complexes.

Analysis of protein conformational characteristics related to thermostability.

The thermal stability of proteins was studied, 195 single amino acid residue replacements reported elsewhere being analysed for several protein conformational characteristics: type of residue replacement; conservative versus nonconservative substitution; replacement being in a homologous stretch of amino acid residues; change in hydrogen bond, van der Waals and secondary structure propensities; solvent-accessible versus inaccessible replacement; type of secondary structure involved in the substitution; the physico-chemical characteristics to which the thermostability enhancement can be attributed; and the relationship of the replacement site to the folding intermediates of the protein, when known. From the above analyses, some general rules arise which suggest where amino acid substitutions can be made to enhance protein thermostability: substitutions are conservative according to the Dayhoff matrix; mainly occur on conserved stretches of residues; preferentially occur on solvent-accessible residues; maintain or enhance the secondary structure propensity upon substitution; contribute to neutralize the dipole moment of the caps of helices and strands; and tend to increase the number of potential hydrogen bonding or van der Waals contacts or improve hydrophobic packing.

Thermal transitions in the structure of tubulin. Environments of aromatic aminoacids.

The environment of aromatic aminoacids in the thermal transition of brain tubulin has been studied by several spectroscopic techniques (Fourth Derivative, Difference Absorption, Fluorescence and Circular Dichroism), in order to study its denaturation. An irreversible, temperature-induced, structural transition was found at around 48 degrees C. In order to establish the relative degree of hydrophobicity of tubulin aromatic residues, before and after the thermal transition, difference and fourth derivative absorption spectra at different temperatures were compared with spectra of tyrosine and tryptophan model compounds in different media. It was found that at high temperatures, tubulin acquires a partially denatured stable state, with a significant amount of residual structure still preserved. This state is characterized by a general increase of the exposure of tyrosine residues to the medium, while the environment of tryptophans becomes more hydrophobic.

Fluorescence study of the effects of histone tyrosyl residues of DNA.

The fluorescence spectra of histone-DNA complexes at pH 7.0 were studied. These species showed fluorescence spectra broader than the characteristic histone spectrum, with the appearance of peaks at higher wavelengths. It is shown that the emission at higher wavelengths arises from the bases of DNA (specially from the A-T bases), when their hydrogen bonds are weakened or broken. These results corroborate those of other authors and it is postulated that tyrosine residues in histone form hydrogen bonds with DNA and/or stack with the bases of the nucleic acid.

Distinctions between mechanisms of cytochalasin D activity for Mg2+- and K+-induced actin assembly.

The fraction of assembled actin and the diffusion coefficients of filamentous and nonfilamentous species have been determined by fluorescence photobleaching recovery. For both Mg2+-induced and K+-induced actin assembly, a higher concentration of cation leads to longer filaments. Cytochalasin D reduces the fraction of actin present as assembled filaments. In the absence of Mg2+, the accompanying increase in diffusion coefficient of the filaments is of an appropriate magnitude to be accounted for by shortening of filaments as a result of net depolymerization. In the presence of Mg2+, cytochalasin D induces a dose-dependent increase in diffusion coefficient up to about a factor of 10. This increase indicates a shortening of filaments consistent with extensive filament cleavage. Under all conditions studied, the unassembled actin is present primarily as monomer.