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Alternating acquisition of background and sample spectra is often employed in conventional Fourier-transform infrared spectroscopy or ultraviolet–visible spectroscopy for accurate background subtraction. For example, for solvent background correction, typically a spectrum of a cuvette with solvent is measured and subtracted from a spectrum of a cuvette with solvent and solute. Ultrafast spectroscopies, though, come with many peculiarities that make the collection of well-matched, subtractable background and sample spectra challenging. Here, we present a demountable split-sample cell in combination with a modified Lissajous scanner to overcome these challenges. It allows for quasi-simultaneous measurements of background and sample spectra, mitigating the effects of drifts of the setup and maintaining the beam and sample geometry when swapping between background and sample measurements. The cell is moving between subsequent laser shots to refresh the excited sample volume. With less than 45 μl of solution for 150 μm optical thickness, sample usage is economical. Cell assembly is a key step and covered in an illustrated protocol.
Vibrational energy transfer (VET) is essential for protein function. It is responsible for efficient energy dissipation in reaction sites, and has been linked to pathways of allosteric communication. While it is understood that VET occurs via backbone as well as via non-covalent contacts, little is known about the competition of these two transport channels, which determines the VET pathways. To tackle this problem, we equipped the β-hairpin fold of a tryptophan zipper with pairs of non-canonical amino acids, one serving as a VET injector and one as a VET sensor in a femtosecond pump probe experiment. Accompanying extensive non-equilibrium molecular dynamics simulations combined with a master equation analysis unravel the VET pathways. Our joint experimental/computational endeavor reveals the efficiency of backbone vs. contact transport, showing that even if cutting short backbone stretches of only 3 to 4 amino acids in a protein, hydrogen bonds are the dominant VET pathway.
Vibrational energy transfer (VET) is emerging as key mechanism for protein functions, possibly playing an important role for energy dissipation, allosteric regulation, and enzyme catalysis. A deep understanding of VET is required to elucidate its role in such processes. Ultrafast VIS-pump/IR-probe spectroscopy can detect pathways of VET in proteins. However, the requirement of having a VET donor and a VET sensor installed simultaneously limits the possible target proteins and sites; to increase their number we compare six IR labels regarding their utility as VET sensors. We compare these labels in terms of their FTIR, and VET signature in VET donor-sensor dipeptides in different solvents. Furthermore, we incorporated four of these labels in PDZ3 to assess their capabilities in more complex systems. Our results show that different IR labels can be used interchangeably, allowing for free choice of the right label depending on the system under investigation and the methods available.