Fluorescence lifetime components reveal kinetic intermediate states upon equilibrium denaturation of carbonic anhydrase II
Methods and Applications in Fluorescence
In most cases, intermediate states of multistage folding proteins are not "visible" under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study intermediate states of carbonic anhydrase II under
... drase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed revealing that the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states. KEYWORDS : carbonic anhydrase II, time-resolved fluorescence spectroscopy, protein intermediate states, comparing of kinetic and equilibrium experiments, protein fluorescence lifetime states with disrupted structure. Experimentalists face such challenge rather frequently. For example, under equilibrium conditions lysozyme, apomyoglobin, ribonuclease A, barnase and some other proteins behave as two-state proteins, and kinetic experiments allow revealing intermediate states formed upon their folding. 7-13 The solution of this problem is to find an experimental method that is sensitive enough to distinguish between different intermediate states of the protein. In this study we have demonstrated that for carbonic anhydrase II the stability of intermediate states upon denaturation can be recorded experimentally by the change in the lifetime components of intrinsic tryptophan fluorescence of this protein. MATERIALS AND METHODS Protein expression and purification. The bovine carbonic anhydrase II coding sequence was reverse-transcribed from total RNA of bone marrow cells. cDNA was synthesized using gene specific oligonucleotide primer 3'-CA2 (5'-tttgtcgacGGCCAGTTCACCAAGTGGACTTGTG-3' (SalI restriction site is underlined) and M-MuLV reverse transcriptase (Fermentas, Lithuania). Products of first strand synthesis were amplified using the polymerase chain reaction and gene specific oligonucleotide primer 5'-CA2 (5'-tacttttcatATGTCCCATCACTGGGGATAC-3') (NdeI restriction site is underlined). The amplified carbonic anhydrase II gene was double digested with SalI and NdeI and inserted into the pET-11c_joe vector between NdeI and SalI restriction sites. The resulting plasmid was designated as pBCAB. Plasmids with the mutant BCA II genes were constructed by a standard PCR technique, using appropriate primers and a pET-28a vector as a template and a QuikChange kit (Stratagene, USA). The DNA sequences of all constructs were confirmed by the DNA sequence analysis. Carbonic anhydrase II and its mutant forms were expressed in Е. coli cells and isolated as described elsewhere. 14, 15 Unfolding and refolding experiments. For refolding studies, samples of unfolded carbonic anhydrase II (solution containing 9.5 M urea, 20 mM phosphate buffer, pH 8) were prepared at high protein concentrations (usually 2-10 mg/ml) and incubated in 9.5 M urea for 24 h in order to reach complete unfolding. Refolding experiments were initiated by 10-to 100-fold dilution of the unfolded protein into the phosphate buffer (20 mM phosphate buffer, pH 8) containing desired urea concentrations. The final protein content in the refolding mixtures varied from 0.01 to 1.0 mg/ml. Experiments on the carbonic anhydrase II unfolding were carried out in a similar manner, by 10-to 100-fold dilution of the native protein to solutions containing desired urea concentrations in 20 mM phosphate buffer, pH 8. Fluorescence unfolding and refolding kinetics were monitored using a Cary Varian Eclipse spectrofluorometer (Agilent Technologies, Australia) equipped with a temperature-controlled holder. The samples were excited at 280 nm and the emission was monitored at 335 nm using the cuvette with 10×10 mm path lengths. Slits of 2 nm were applied for excitation and emission. All measurements were performed in 20 mM phosphate buffer at pH 8, 20C. All obtained kinetic curves were well fitted by a double-exponential approximation. Kinetic data were repeatedly obtained five times for each point. Kinetic experiments were performed in the absence of zinc which allowed excluding the slow phase of zinc binding detected only upon measuring the kinetics of folding. Steady-state and time-resolved fluorescence measurements. Steady-state and time-resolved fluorescence measurements were performed using Fluorolog 3-22 spectrofluorometer (Horiba Jobin Yvon, USA) equipped with DeltaHub timing module for the time-correlated single photon counting. For steady-state experiments 450 W Xenon arc lamp was used as an excitation source, for time-resolved NanoLED pulsed diode (296 nm, a pulse duration of ~ 1.2 ns) was applied. Steady-state fluorescence spectra were corrected for the background intensities and for the inner filter effect. Time-resolved fluorescence decays were collected in the range of 305-419 nm with increment of 3 nm and a time resolution of 27 ps/channel. The UV-Vis absorption spectra were measured with a Cary 5000i spectrophotometer (Agilent Technologies, Australia). The final protein concentration (6.6 µM) was calculated from absorption at 280 nm using the extinction coefficient 54.5·10 3 M −1 cm −1 . Equilibrium denaturation was performed by incubating the proteins during not less than 12 hours at 20C in 20 mM Tris-HCl buffer, pH 8.2, containing increasing concentrations of urea (0-7.5 M). The final concentration of urea was defined from refractive index measured by IRF-454-B2 M (KOMZ, Kazan, Russia).