Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the β2-adrenergic receptor using ~109-fold less protein than conventional assays.

Allosteric regulation of enzymatic activity forms the basis for controlling a plethora of vital cellular processes. While the mechanism underlying regulation of multimeric enzymes is generally well understood and proposed to primarily operate via conformational selection, the mechanism underlying allosteric regulation of monomeric enzymes is poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (Thermomyces lanuginosus Lipase) while titrating its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate with the enzyme's major conformational states and are redistributed in the presence of the regulatory effector. Thus, our data support allosteric regulation of monomeric enzymes to operate via selection of preexisting functional states and not via induction of new ones.

Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored. Here we present a single-enzyme study that reveals that cytochrome bo3 from Escherichia coli, an HCO closely homologous to Complex IV in human mitochondria, can enter a rare, long-lifetime leak state during which proton flow is reversed. The probability of entering the leak state is increased at higher ΔpH. By rapidly dissipating the PMF, we propose that this leak state may enable cytochrome bo3, and possibly other HCOs, to maintain a suitable ΔpH under extreme redox conditions.

The advent of advanced single molecule measurements heralded the arrival of a wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways not deducible by conventional bulk assays. They offered the direct observation and quantification of the abundance and life time of multiple states and transient intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements, thus providing unprecedented insights into complex biological processes. Here we survey the current state of the art in single-molecule fluorescence microscopy methodology for studying the mechanism of enzymatic activity and the insights on protein functional dynamics. We will initially discuss the strategies employed to date, their limitations and possible ways to overcome them, and finally how single enzyme kinetics can advance our understanding on mechanisms underlying function and regulation of proteins. [Formula: see text]Special Issue Comment: This review focuses on functional dynamics of individual enzymes and is related to the review on ion channels by Lu,44 the reviews on mathematical treatment of Flomenbom45 and Sach et al.,46 and review on FRET by Ruedas-Rama et al.41

G-Protein Coupled Receptors (GPCRs) are structurally flexible membrane proteins(1), that mediate a host of physiological responses to extracellular ligands like hormones and neurotransmitters(2). Details of the dynamic structural behavior are hypothesized to encode functional plasticity seen in GPCR activity(1), where ligands with different efficacies can direct the same receptor towards different signaling phenotypes. Although the number of GPCR crystal structures is increasing(3-5), the receptors are characterized by complex and poorly understood conformational landscapes(6). Therefore, we have developed a fluorescence microscopy assay to study the activation and dynamics of single b2-Adrenergic Receptors (b2ARs) reconstituted in liposomes. Conformational fluctuations are monitored by changes in intensity of a small fluorescent molecule conjugated to an endogenous cysteine located at the cytoplasmic end of the sixth trans-membrane helix of the receptor. By imaging arrays of surface-tethered proteoliposomes, we can read out the dynamic properties of hundreds of single b2AR reconstituted in a lipid membrane. Our data reveal subtle changes in b2AR conformational dynamics with agonist stimulation, which would be undetectable in bulk assays(7-9).

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Bacterial flagellum is a large complex organelle made up of more than 30 different proteins. Most of the flagellar proteins are localized outside of the cell and are exported across the cytoplasmic membrane by the flagellar type III secretion system. The protein export is highly regulated. Membrane protein FlhB plays a key role in this regulation. The protein consists of two domains: a hydrophobic N-terminal part (FlhB TM), which is predicted to have four transmembrane helices, and a C-terminal cytoplasmic domain (FlhB C). Homologues of FlhB were found in all bacterial type III secretion systems. Sequences of these proteins are highly conserved suggesting that their function is also similar. In this study we have compared properties of FlhB from two organisms: Salmonella typhimurium and Aquifex aeolicus. Salmonella and Aquifex FlhB share 32% sequence identity. However, these proteins are evolutionarily very distant. Comparison of the two proteins may provide us with additional information about functionally important regions of FlhB. We have substituted flhB gene in Salmonella by flhB of A. aeolicus or by chimera gene encoding hybrid FlhB composed of the FlhB TM of S. typhimurium and FlhB C of A. aeolicus. Then we analyzed motility of the mutants on soft agar plates. Although all mutants showed some motility, they were substantially less motile than wild-type cells. We have found several spontaneous mutations in C-terminal part of FlhB that resulted in enhanced motility. To understand the effect of the mutations we have solved FlhB C structures from both organisms: Salmonella and Aquifex. We have also determined secondary structure and stability of the mutated FlhB C. Based on our findings we suggest that conformational flexibility is important for FlhB function.

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