The heat released during catalytic turnover enhances the diffusion of an enzyme

Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis,. Although this observation has been reported and characterized for several different systems–, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms,. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein–solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme

An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational phenotypes of interest

Nonequilibrium Single Molecule Protein Folding in a Coaxial Mixer

We have developed a continuous-flow mixing device suitable for monitoring bioconformational reactions at the single-molecule level with a response time of ∼10 ms under single-molecule flow conditions. Its coaxial geometry allows three-dimensional hydrodynamic focusing of sample fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. The capillary-based design enables rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each device to many months. In this article, to determine both the distance-to-time transfer function and the instrument response function of the device we characterize its fluid flow and mixing properties using both fluorescence cross-correlation spectroscopy velocimetry and finite element fluid dynamics simulations. We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By transiently populating the unfolded state of Chymotrypsin Inhibitor Protein 2 (CI2) under nonequilibrium in vitro refolding conditions, we spatially and temporally resolve the denaturant-dependent nonspecific collapse of the unfolded state from the barrier-limited folding transition of CI2. Our results are consistent with previous CI2 mixing results that found evidence for a heterogeneous unfolded state consisting of cis- and trans-proline conformers