Sizing up single-molecule enzymatic conformational dynamics

Abstract

Enzymatic reactions and related protein conformational dynamics are complex and inhomogeneous, playing crucial roles in biological functions. The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. It is extremely difficult to characterize and analyze such complex dynamics in an ensemble-averaged measurement, especially when the enzymes are associated with multiple-step, multiple-conformation complex chemical interactions and transformations. Beyond the conventional ensemble-averaged studies, real-time single-molecule approaches have been demonstrated to be powerful in dissecting the complex enzymatic reaction dynamics and related conformational dynamics. Single-molecule enzymology has come a long way since the early demonstrations of the single-molecule spectroscopy studies of enzymatic dynamics about two decades ago. The rapid development of this fundamental protein dynamics field is hand-in-hand with the new development of single-molecule imaging and spectroscopic technology and methodology, theoretical model analyses, and correlations with biological preparation and characterization of the enzyme protein systems. The complex enzymatic reactions can now be studied one molecule at a time under physiological conditions. Most exciting developments include active manipulation of enzymatic conformational changes and energy landscape to regulate and manipulate the enzymatic reactivity and associated conformational dynamics, and the new advancements have established a new stage for studying complex protein dynamics beyond by simply observing but by actively manipulating and observing the enzymatic dynamics at the single-molecule sensitivity temporally and spatially.

Protein Conformational Landscapes and Catalysis. Influence of Active Site Conformations in the Reaction Catalyzed by L-Lactate Dehydrogenase

“…4,5 Moreover, the presence of different conformations in any of the states along the full catalytic process is essential to explain experimental results such as the temperature dependence of kinetic isotope effects (KIEs) 6 , 7 or the significant different rate constants observed in single-molecule spectroscopy. 8 In this regard, Yeung and co-workers monitored differences in the chemical reactivity of individual Lactate Dehydrogenase (LDH) molecules, finding that the activity of individual enzyme molecules could vary by a factor of three or four. 9 This pioneering single molecule-experiment on LDH have been recently supported by temperature jump infrared spectroscopy studies performed by Dyer and co-workers, 10 revealing the presence of multiple reactive conformations of pyruvate bound to the enzyme, each of them forming a heterogeneous branched reaction pathway with a different rate of pyruvate to lactate conversion.…”

Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits

“…As reported here, such information can be elucidated by single-molecule techniques; observation of intermediate steps and transition states are otherwise lost through averaging in ensemble populations. 22,23 DNA polymerization experiments employing single-molecule Forster resonance energy transfer (smFRET) have revealed conformational flexibility and insights into the fidelity mechanism of KF. 24–27 Additionally, the observed kinetic and thermodynamic differences between correct and mismatched dNTP-DNA-polymerase complexes quantify the driving forces for correct nucleotide incorporation into DNA templates.…”

Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

“…In modern enzymology, it has extensively been explored that the enzymatic conformation-dynamics-function relationship, especially the dynamic rather than the static perspectives, plays a critical role in the understanding of enzyme mechanisms at the molecular level (18)(19)(20)(21). For example, in an enzymatic reaction, formation of the enzyme-substrate complex often involves significant enzymatic active site conformational changes (22)(23)(24)(25).…”

“…For example, the studies of enzymatic stability focused on ensemble level activity of enzymes at different physical conditions or chemical environment without probing corresponding change in conformation of enzyme molecules under the same conditions (11,12,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). In recent years, a number of technical approaches on single-molecule protein conformational manipulation, such as atomic force microscopy (AFM), magnetic tweezers, and optical tweezers, have been developed (29)(30)(31)(32)(33)(34).…”