Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

Abstract

2D-IR spectroscopy has matured to a powerful technique to study the structure and dynamics of peptides, but its extension to larger proteins is still in its infancy, the major limitations being sensitivity and selectivity. Site-selective information requires measuring single vibrational probes at sub-millimolar concentrations where most proteins are still stable, which is a severe challenge for conventional (FT)IR spectroscopy. Besides its ultrafast time-resolution, a so far largely underappreciated potential of 2D-IR spectroscopy lies in its sensitivity gain. The present paper sets the goals and outlines strategies how to use that sensitivity gain together with properly designed vibrational labels to make IR spectroscopy a versatile tool to study a wide class of proteins. Abstract Abstract: 2D-IR spectroscopy has matured to a powerful technique to study the structure

Intrinsic phasing of heterodyne-detected multidimensional infrared spectra

“…The boxcar geometry, however, still yields the highest possible sensitivity for the collection of multidimensional IR spectra, and in extending the technique to ever lower concentrations and to weaker absorbers [24], methods to enhance the reliability of phasing and ease of use of boxcar geometry 2D IR are still of great importance. The present phasing methodology based on analysis of the heterodyne-detected signals themselves thus represent an important first step in bringing the ease of use of pump-probe geometry 2D IR to boxcar geometry spectrometers.…”

“…4(a)). The azido band is sitting on a broad background [14,24] originating from the excited state absorption of the OD stretch vibration of the D 2 O buffer, where the fundamental of this transition can still be excited in the very red wing of the band [25] with the pump pulses, centered at 2110 cm −1 , used for this experiment. The associated phase response (Fig.…”

Perspective: Chain dynamics of unfolded and intrinsically disordered proteins from nanosecond fluorescence correlation spectroscopy combined with single-molecule FRET

“…Infrared spectroscopy is starting to resolve site-specific information down to picosecond times for larger proteins. 119 Neutron spin echo spectroscopy 120 may be the only other method besides nsFCS currently available for probing (bio)polymer chain dynamics in the tens of nanosecond range; it can reveal a large spectrum of relaxation modes and has been used to identify internal friction. 121,122 Arguably the most popular technique for IDPs is nuclear magnetic resonance (NMR) spectroscopy: While the time scales of global chain dynamics are difficult to access with NMR because of rotational decorrelation, the technique is ideal for obtaining a wealth of information on local dynamics, especially on the level of individual residues or segments forming secondary structures.…”

Ultrafast dynamics induced by the interaction of molecules with electromagnetic fields: Several quantum, semiclassical, and classical approaches

“…Benzonitrile (PhCN) is another potentially useful probe to determine the local electrostatic environment as it fulfills three important criteria: the -CN stretching mode at $\approx 2200$ cm – 1 (a) absorbs in a frequency range (i.e., between 1800 cm – 1 and 2800 cm – 1 ) in which proteins containing only naturally occurring amino acids have no vibrational spectral response (except for the -SH group in cysteine), (b) is to a good approximation a local mode (i.e., uncoupled from other framework modes), and (c) the dipole moment of PhCN is to a large extent that of the nitrile group itself. On the other hand, the nitrile group may pose additional challenges in concrete experiments due to its low extinction coefficient 293 . The previous work on PhCN in water 294 provides an ideal benchmark to validate the computational methods used in the present work.…”