Temperature-Jump 2D IR Spectroscopy with Intensity-Modulated CW Optical Heating

Abstract

Pulsed temperature-jump (T-jump) spectroscopy with infrared (IR) detection has been widely used to study biophysical processes occurring from nanoseconds to ~1 millisecond with structural sensitivity. However, many systems exhibit structural dynamics on timescales longer than the millisecond barrier that is set by the time-scale for thermal relaxation of the sample. We have developed a linear and nonlinear infrared spectrometer coupled to an intensity-modulated continuous wave (CW) laser to probe T-jump initiated chemical reactions from <1 ms to seconds. Time-dependent modulation of the CW laser leads to a <1 ms heating time as well as a constant final temperature (± 3%) for the duration of the heating time. Temperature changes of up to 75 °C in D 2 O are demonstrated, allowing for nonequilibrium measurements inaccessible to standard pulsed optical T-jump setups. T-jump linear absorption, pump-probe, and two-dimensional IR (2D IR) spectroscopy are applied to the unfolding and refolding of ubiquitin and a model intercalated motif (i-motif) DNA sequence, and analysis of the observed signals is used to demonstrate the limits and utility of each method. Overall, the ability to probe temperature-induced chemical processes from <1 ms to many seconds with 2D IR spectroscopy provides multiple new avenues for time-dependent spectroscopy in chemistry and biophysics.

Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism

“…27,28 To extend the observation time window up to seconds, CW infrared (IR) excitation sources have been used in combination with single-molecule spectroscopy, microfluidics, and very recently 2D IR spectroscopy. 22,24,25 In a similar way, we implemented a 5 W CW laser diode emitting at 1.5 μm, for the sample excitation, in our T-jump setup. 23 The IR beam was focused onto a 150 × 140 μm 2 spot in a 100 μm optical path sample cell to ensure the homogeneous heating and cooling of the sample by keeping a weak absorption of the solvent.…”

“…Sample cooling is slightly slower and displays a biexponential decay with the time constants of 30 ± 2.2 ms (82%) and 1.7 s (18%), respectively. Such multiphasic changes of the temperature has been already observed under similar experimental conditions and discussed in detail in ref 22. Importantly, as previously reported, the heating and cooling dynamics are found to be independent of the initial temperature and the T-jump amplitude (for details see the Supporting Information).…”

“…Unlike short DNA duplexes, the melting dynamics of short intramolecular G4s are expected to occur on longer time scales, , making pulsed laser T-jump initiation inappropriate for probing their dissociation. In this regard, recent developments have been achieved for the use of CW T-jump initiation to access both the denaturation and the renaturation dynamics of biomolecules beyond the millisecond time scale. − Herein, we present the first study of this type, devoted to G4-forming sequences. We compare the thermal denaturation and renaturation dynamics of four G4-forming sequences: three antiparallel topologies made of the human telomeric sequences Tel21 (d­[(5′-GGG­(TTAGGG) 3 -3′]) and Tel22 (d­[5′-AGGG­(TTAGGG) 3 -3′]) and the short thrombin binding aptamer sequence (d­[5′-GGTTGG­TGTGGTTGG-3′]) and the parallel topology made of the human CEB25 minisatellite sequence 26CEB (d­[5′-AAGGGTG­GGTGTAAGT­GTGGGTGGGT-3′]).…”

Resolving Dynamics in the Ensemble: Finding Paths through Intermediate States and Disordered Protein Structures

“…To leverage this general physical property, the temperature-jump (T-jump), usually induced by a laser pulse that excites the overtone stretch mode of water in aqueous solution, can trigger a temperature increase within nanoseconds that lasts up to milliseconds. 4,22,64 This indirect light activated pump−probe approach has been widely used to observe disordered protein regions and oligomerization mechanisms. 4,22 To utilize computational refinements for time-resolved processes requires both a time-resolved experimental input and MD trajectories to efficiently sample structures away from the stationary state.…”

“…Many proteins, photoactive or not, populate non-native states strongly correlated to the surrounding temperature. To leverage this general physical property, the temperature-jump ( T -jump), usually induced by a laser pulse that excites the overtone stretch mode of water in aqueous solution, can trigger a temperature increase within nanoseconds that lasts up to milliseconds. ,, This indirect light activated pump–probe approach has been widely used to observe disordered protein regions and oligomerization mechanisms. , …”