Chaperone-like activity of tubulin

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 β- and γ-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

Refolding of urea-denatured tubulin: recovery of nativelike structure and colchicine binding activity from partly unfolded states

Tubulin unfolding in urea proceeds via the formation of a partially unfolded intermediate state, stable in 2 M urea, that unfolds further in higher urea concentrations. The intermediate state had spectroscopic properties reminiscent of a molten globule and negligible colchicine binding activity. Refolding of totally unfolded tubulin in 8 M urea yielded an intermediatelike state characterized by partial burial of tryptophans and partial recovery of secondary and tertiary structures, although colchicine-binding activity of the protein was not regained. Further folding of this intermediatelike state, toward the native conformation, with respect to both structural and functional parameters did not occur. However, a significant percentage of colchicine binding activity and nativelike tertiary structure was recovered when refolding was initiated from partially denatured protein samples, viz., from <1.2 M urea. Thus, although high concentration of urea induced loss of structure and activity was irreversible, the conformational changes induced in restricted regions of tubulin by lower concentrations of urea, which are probably crucial for its various functional properties, could be reversed

A partially folded intermediate during tubulin unfolding: its detection and spectroscopic characterization

The unfolding reaction of the dimeric protein tubulin, isolated from goat brain, was studied using fluorescence and circular dichroism techniques. The unfolding of the tubulin dimer was found to be a two-step process at pH 7. The first step leads to the formation of an intermediate conformation, stable at around 1-2 M urea, followed by a second step that was due to unfolding of the intermediate state. At pH 3, the urea-induced biphasic unfolding profiles obtained at pH 7 became a one-step process indicating that a stable intermediate was also formed at this pH. The intermediate at pH 3 was more stable toward urea denaturation than that at pH 7. The intermediate state has about 60% secondary structure, partially exposed aromatic residues, and less tertiary structure as compared to the native states. Also, hydrophobic surfaces were more exposed in the intermediate than in the native or unfolded states. These results indicate that the intermediate state observed during tubulin unfolding is not only distinct from both the native and unfolded forms but also possesses some properties characteristic of a molten globule

Tubulin conformation and dynamics: a red edge excitation shift study

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 β-subunit of the tubulin dimer, probably makes contact with the a-subunit of tubulin and imparts a rigidity to that region of the protein, which facilitates the REES

Differential interactions of the Mg²⁺ complexes of chromomycin A₃ and mithramycin with Poly(dG-dC)•Poly(dC-dG) and Poly(dG)•Poly(dC)

The interaction of the two anticancer antibiotics, chromomycin A3 and mithramycin, with the polynucleotides poly(dG-dC).poly(dC-dG), representative of B-DNA, and poly(dG)•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•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•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•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

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 β-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p. In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin. GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability. These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes

Retraction to: The EMBO Journal (2003) 22, 5230–5240, doi:10.1093/emboj/cdg48