RESUMO
The folding/unfolding equilibrium is investigated in poly(glutamic acid) (PGA) by two complementary sets of experiments: temperature-dependent steady-state circular dichroism spectra on the one hand and time-resolved circular dichroism measurements coupled with a T-jump experiment on the other hand. The experiments are performed for PGA dissolved in water for various pH values, as well as in heavy water. The kinetic and thermodynamic parameters extracted from these measurements are shown to be markedly different between light and heavy water, which is assigned to the difference in hydrogen bond energies in both solvents.
Assuntos
Óxido de Deutério/química , Ácido Poliglutâmico/química , Água/química , Dicroísmo Circular , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Dobramento de Proteína , Desdobramento de Proteína , Temperatura , TermodinâmicaRESUMO
Motions of the trans-p-coumaric acid carbonyl group following the photoexcitation of the R52Q mutant of photoactive yellow protein (PYP) are investigated, for the first time, by ultrafast time-resolved circular dichroism (TRCD) spectroscopy. TRCD is monitored in the near-ultraviolet, over a time scale of 10 ps. Immediately after excitation, TRCD is found to exhibit a large negative peak, which decays within a few picoseconds. A quantitative analysis of the signals shows that, upon excitation, the carbonyl group undergoes a fast (âª0.8 ps) and unidirectional flipping motion in the excited state with an angle of ca. 17-53°. For the subset of proteins that do not enter the signaling photocycle, TRCD provides strong evidence that the carbonyl group moves back to its initial position, leading to the formation of a nonreactive ground-state intermediate of trans conformation. The initial ground state is then restored within ca. 3 ps. Comparative study of R52Q and wild-type PYP provides direct evidence that the absence of Arg52 has no effect on the conformational changes of the chromophore during those steps.
Assuntos
Proteínas de Bactérias/química , Ácidos Cumáricos/química , Halorhodospira halophila/química , Fotorreceptores Microbianos/química , Proteínas de Bactérias/genética , Dicroísmo Circular , Halorhodospira halophila/genética , Processos Fotoquímicos , Fotorreceptores Microbianos/genética , Mutação Puntual , PropionatosRESUMO
Poly(glutamic acid) has been studied with a nanosecond T-jump experiment. A new experimental set-up based on the frequency-quadrupling of an 82 MHz Titanium-Sapphire laser allows rapid CD measurements to be performed. Combining time-resolved absorption and circular dichroism at 204 and 220 nm, we are able to measure precisely the unfolding relaxation time as well as the helical fraction evolution. We show that only CD at 220 nm is relevant to observe the unfolding of an alpha helix whereas no change is observed for CD at 204 nm. Conversely, both absorptions yield information on the dynamics of the process.
Assuntos
Dicroísmo Circular/métodos , Ácido Glutâmico/química , Dobramento de Proteína , Óxido de Alumínio/química , Cinética , Lasers , Polímeros/química , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Titânio/químicaRESUMO
The use of a fast temperature jump (T-jump) is a very powerful experiment aiming at studying protein denaturation dynamics. However, probing the secondary structure is a difficult challenge and rarely yields quantitative values. We present the technical implementation of far-UV circular dichroism in a nanosecond T-jump experiment and show that this experiment allows us to follow quantitatively the change in the helical fraction of a poly(glutamic acid) peptide during its thermal denaturation with 12 ns time resolution.
