Infrared and Fluorescence Assessment of Protein Dynamics: From Folding to Function

Abstract

While folding or performing functions, a protein can sample a rich set of conformational space. However, experimentally capturing all of the important motions with sufficient detail to allow a mechanistic description of their dynamics is nontrivial since such conformational events often occur over a wide range of time and length scales. Therefore, many methods have been employed to assess protein conformational dynamics and, depending on the nature of the conformational transition in question, some may be more advantageous than others. Herein, we describe our recent efforts, and also those of others, wherever appropriate, to use infrared- and fluorescence-based techniques to interrogate protein folding and functional dynamics. Specifically, we focus on discussing how to use extrinsic spectroscopic probes to enhance the structural resolution of these techniques and how to exploit various cross-linking strategies to acquire dynamic and mechanistic information that was previously difficult to attain.

Fermi resonance as a means to determine the hydrogen-bonding status of two infrared probes

“…2D IR spectra were collected on a photon-echo setup with a boxcar geometry, the details of which have been described in detail elsewhere. 24 IR transient kinetics were obtained on a home-built, 1 kHz pump-probe apparatus, where the pump pulse (3 μJ) and probe pulse (0.2 μJ) were derived from the same mid-IR pulse (~120 fs and ~150 cm −1 bandwidth) with a center frequency of 2230 cm −1 (1740 cm −1 ) for 4-CI (CP). After spatial overlap of the pump and probe pulses in the sample, the relative time delay between which was controlled by an optical delay line, the probe beam was directed to a monochromator and dispersed onto a 32 channel mercury cadmium telluride (MCT) array detector (Infrared Systems Development, Winter Park, FL).…”

Activation pH and Gating Dynamics of Influenza A M2 Proton Channel Revealed by Single‐Molecule Spectroscopy

“…Because this quenching effect arises from a photoinduced electron transfer (PET) process, it requires the quencher (Trp) to be sufficiently close to the fluorophore (dye). In addition, as the rate of PET is dependent on the quencher–fluorophore separation distance ( R QF ), it is possible to utilize a Trp–dye pair and fluorescence correlation spectroscopy (FCS) to characterize the rate of a specific protein conformational motion, as long as this motion modulates R QF and hence induces fluctuations in the fluorescence intensity . To employ this PET‐FCS strategy to assess the conformational dynamics of Trp41 in the full‐length M2 and M2TM proton channels, we introduced an Atto 655 dye at position 40 through a Cys residue using a double mutant of the respective native sequence, Leu40/Cys‐Trp41/Phe (the labeled peptide and protein are hereafter referred to as M2TM* and M2*, respectively).…”

Simple method to introduce an ester infrared probe into proteins

“…Infrared (IR) spectroscopy is a well‐established and widely used technique for studying protein structure, dynamics and function . This is because a protein's backbone and sidechains can give rise to a multitude of vibrational transitions, some of which are sensitive to factors that depend on either environment, conformation, or both .…”

“…Infrared (IR) spectroscopy is a well-established and widely used technique for studying protein structure, dynamics and function. 1,2 This is because a protein's backbone and sidechains can give rise to a multitude of vibrational transitions, some of which are sensitive to factors that depend on either environment, conformation, or both. 3 For example, the amide I 0 band arising from the backbone carbonyls has been extensively used to assess protein folding dynamics and mechanisms due to its sensitivity to protein secondary structures.…”