that is close to unfolding transition state (Fig. 24b). To be able to reach the
unfolding transition state, the sample needs to be pre-heated to a temperature of
63°C. This pre-heating of the sample is necessary since comparable T-jumps do not
lead to unfolding of the protein at lower temperatures. The T-jump induces a barrier
shift towards the folded state (F), leading to a downhill unfolding (U) of a part of the
sample (A in Fig. 24b). Afterwards, a remaining sub-ensemble can unfold by
overcoming the shifted energy barrier on a longer timescale (B in Fig. 24b).
Figure 24c shows the corresponding 2D IR spectrum at elevated temperature in the
amide-I spectral range, which is characterized by intense diagonal peaks and weak
cross peaks (arrows). The two beta-sheet modes lie at positions of 1642 cm
-1 (m \ )
and blue 1676 cm
-1 (m k ), respectively. A change in the stationary sample
temperature (Fig. 24d) leads to disruption of the beta-sheet of ubiquitin. This is
observed by a blue-shift of the m \ mode, which can be best visualized in a difference
2D IR signal between the two temperatures (red arrows in Fig. 24e). The blue and
green arrows indicate an increase in the random-coil structure and a depletion of the
cross peak signals, respectively. Figure 24f–k demonstrates, how the accompanied
structural changes in the molecules can be tracked with transient 2D IR T-jump
spectroscopy. At short T-jump delays (100 ns), the T-jump difference spectrum
shows predominately temperature-induced disruption of H-bonds (red ellipse),
which cause a blue-shift of the sheet vibrations. Following that initial response, the
difference spectra gradually evolve up to delays as large as 7 ms, (g)–(k), where
almost a quantitative agreement with the stationary difference spectrum, (e), is
obtained. Through a kinetic analysis of a full series of such T-jump 2D IR spectra,
as well as a comparison with MD simulations, it was possible to obtain a detailed
understanding of the progressive unfolding mechanism of ubiquitin. It was revealed
that ubiquitin unfolds in three distinguishable phases, i.e. a first stage of thermal
excitation of the solvent, a second stage of microsecond downhill unfolding, and a
third stage of ms-unfolding over a barrier (Fig. 24b). Based on the close matching of
experimental and MD simulation results it was furthermore proposed that the
T-jump 2D IR spectroscopy can be used to discriminate between different
theoretically proposed unfolding pathways.
T-jump 2D IR spectroscopy has also been used to study structural changes of
other biological sample systems. Also, Tokmakoff et al. have studied the folding
mechanism of the N-terminal domain of ribosomal proteins and compared the
experimental results to Markov-state based MD-simulations [262]. Native and
denaturized structures could be correctly predicted and time-constants for the
temperature-induced interconversion were successfully modeled. Other applications
by the same group considered protein-protein binding and T-jump induced
dissociation dynamics of insulin homodimers [271]. A detailed comparison of the
observable kinetics at different starting temperatures indicated that the dissociation
is characterized by two processes, the influence of which varies with temperature.
Future applications of T-jump 2D IR spectroscopy are moreover likely to involve
combinations of T-jump 2D IR spectroscopy with other established methods such
isotope labelling and site-specific mutations. Finally, additional future experiments
may involve addressing distinguishable conformational states in the folded and
unfolded states of various proteins, as well timescales of exchange between them.
Top Curr Chem (Z) (2017) 375:86
123
171
Reprinted from the journal
unfolding transition state, the sample needs to be pre-heated to a temperature of
63°C. This pre-heating of the sample is necessary since comparable T-jumps do not
lead to unfolding of the protein at lower temperatures. The T-jump induces a barrier
shift towards the folded state (F), leading to a downhill unfolding (U) of a part of the
sample (A in Fig. 24b). Afterwards, a remaining sub-ensemble can unfold by
overcoming the shifted energy barrier on a longer timescale (B in Fig. 24b).
Figure 24c shows the corresponding 2D IR spectrum at elevated temperature in the
amide-I spectral range, which is characterized by intense diagonal peaks and weak
cross peaks (arrows). The two beta-sheet modes lie at positions of 1642 cm
-1 (m \ )
and blue 1676 cm
-1 (m k ), respectively. A change in the stationary sample
temperature (Fig. 24d) leads to disruption of the beta-sheet of ubiquitin. This is
observed by a blue-shift of the m \ mode, which can be best visualized in a difference
2D IR signal between the two temperatures (red arrows in Fig. 24e). The blue and
green arrows indicate an increase in the random-coil structure and a depletion of the
cross peak signals, respectively. Figure 24f–k demonstrates, how the accompanied
structural changes in the molecules can be tracked with transient 2D IR T-jump
spectroscopy. At short T-jump delays (100 ns), the T-jump difference spectrum
shows predominately temperature-induced disruption of H-bonds (red ellipse),
which cause a blue-shift of the sheet vibrations. Following that initial response, the
difference spectra gradually evolve up to delays as large as 7 ms, (g)–(k), where
almost a quantitative agreement with the stationary difference spectrum, (e), is
obtained. Through a kinetic analysis of a full series of such T-jump 2D IR spectra,
as well as a comparison with MD simulations, it was possible to obtain a detailed
understanding of the progressive unfolding mechanism of ubiquitin. It was revealed
that ubiquitin unfolds in three distinguishable phases, i.e. a first stage of thermal
excitation of the solvent, a second stage of microsecond downhill unfolding, and a
third stage of ms-unfolding over a barrier (Fig. 24b). Based on the close matching of
experimental and MD simulation results it was furthermore proposed that the
T-jump 2D IR spectroscopy can be used to discriminate between different
theoretically proposed unfolding pathways.
T-jump 2D IR spectroscopy has also been used to study structural changes of
other biological sample systems. Also, Tokmakoff et al. have studied the folding
mechanism of the N-terminal domain of ribosomal proteins and compared the
experimental results to Markov-state based MD-simulations [262]. Native and
denaturized structures could be correctly predicted and time-constants for the
temperature-induced interconversion were successfully modeled. Other applications
by the same group considered protein-protein binding and T-jump induced
dissociation dynamics of insulin homodimers [271]. A detailed comparison of the
observable kinetics at different starting temperatures indicated that the dissociation
is characterized by two processes, the influence of which varies with temperature.
Future applications of T-jump 2D IR spectroscopy are moreover likely to involve
combinations of T-jump 2D IR spectroscopy with other established methods such
isotope labelling and site-specific mutations. Finally, additional future experiments
may involve addressing distinguishable conformational states in the folded and
unfolded states of various proteins, as well timescales of exchange between them.
Top Curr Chem (Z) (2017) 375:86
123
171
Reprinted from the journal