Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.

The fluorescence emission maximum of a polar fluorophore in viscous medium often shows a dependence on excitation wavelength, a phenomenon which is named red edge excitation shift (REES). We have found that the fluorescence spectra of the tubulin tryptophans exhibit a REES of about 7 nm. Also, their steady state fluorescence polarization and mean lifetimes show a dependence on both excitation and emission wavelengths. These results indicate that the average tryptophan environment in tubulin is motionally restricted. Although the tryptophan(s) responsible for the observed REES effect could not be localized, it could be concluded from energy transfer experiments with the tubulin-colchicine complex that the tryptophan(s) participating in energy transfer with bound colchicine probably does not contribute to the REES. A REES of 7 nm was also observed in the case of colchicine complexed with tubulin. However, such a REES was not seen in similar studies with the B-ring analogs of colchicine, viz. 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (called AC because it lacks the B ring of colchicine) and deacetamidocolchicine (which lacks the acetamido substituent at the C-7 position of the B ring). There may be two possible reasons to explain these data. (1) Structural differences between colchicine and its analogs may give rise to differences in their excited state dipole moments which will directly affect the extent of REES, and (2) The B-ring substituent, hanging outside the colchicine binding site on the beta-subunit of the tubulin dimer, probably makes contact with the alpha-subunit of tubulin and imparts a rigidity to that region of the protein, which facilitates the REES.

The interaction of the two anticancer antibiotics, chromomycin A3 and mithramycin, with the polynucleotides poly(dG-dC) x poly(dC-dG), representative of B-DNA, and poly(dG) x poly(dC), representative of A-DNA, in the presence of Mg2+ is studied by spectroscopic techniques such as absorbance, fluorescence, and dircular dichroism (CD). The studies were done with both drug x Mg2+ complexes, I and II, having 1:1 and 2:1 stoichiometries with respect to drug and Mg2+, respectively [Aich, P., Sen, R., & Dasgupta, D. (1992) Biochemistry 31, 2988-2997]. The objective of the present work is 2-fold. First, an attempt is made to understand the structural basis of the ligand-DNA interaction, particularly the role of DNA backbone conformation with its groove size and the accessibility of the 2-amino group in the minor groove of guanosine. Second, the role of the antibiotic saccharide moieties in the association with DNA was studied. For this purpose, the spectroscopic characterization of the binding was done followed by the evaluation of binding parameters and associated thermodynamics. Analysis of the observed thermodynamics for the ligand-DNA interactions in terms of the different structures of the polynucleotides was done. The salient results are as follows. Complex I does not discriminate significantly among the A- and B-forms of DNA when it binds to them in an entropy-driven process. On the other hand, complex II for both drugs recognizes B- and A-forms of DNA in different ways. This observation implies that the sequence specificity shown by this complex is a sequel to the difference in the parameters such as groove size and accessibility of the guanosine amino group. Another important finding is that binding with the same polynucleotide is not comparable for the complex II of the two drugs. It emphasizes the involvement of the sugar moieties, when the drug x Mg2+ complex binds to DNA. The presence of an acetoxy group in the sugars of chromomycin A3 imparts some distinctive specific features of the association of the chromomycin dimer x Mg2+ complex with DNA. Finally, the results are compared with those available from NMR studies of different drug-oligonucleotide complexes under conditions where complex II is the ligand.

Tubulin, a ubiquitous protein of eukaryotic cytoskeleton, is a building block unit of microtubule. Although several cellular processes are known to be mediated through the tubulin-microtubule system, the participation of tubulin or microtubule in protein folding pathway has not yet been reported. Here we show that goat brain tubulin has some functions and features similar to many known molecular chaperones. Substoichiometric amounts of tubulin can suppress the non-thermal and thermal aggregation of a number of unrelated proteins such as insulin, equine liver alcohol dehydrogenase, and soluble eye lens proteins containing ␤-and ␥-crystallins. This chaperone-like activity of tubulin becomes more pronounced as temperature increases. Aging of tubulin solution at 37°C also enhances its chaperone-like activity. Tubulin loses its chaperone-like activity upon removal of its flexible hydrophilic C-terminal tail. These results suggest that both electrostatic and hydrophobic interactions are important in substrate binding by tubulin and that the negatively charged C-terminal tails play a crucial role for its chaperone-like activity.