Laboratory diagnosis of disorders of peroxisomal biogenesis and function: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Peroxisomal disorders are a clinically and genetically heterogeneous group of diseases caused by defects in peroxisomal biogenesis or function, usually impairing several metabolic pathways. Peroxisomal disorders are rare; however, the incidence may be underestimated due to the broad spectrum of clinical presentations. The inclusion of X-linked adrenoleukodystrophy to the Recommended Uniform Screening Panel for newborn screening programs in the United States may increase detection of this and other peroxisomal disorders. The current diagnostic approach relies heavily on biochemical genetic tests measuring peroxisomal metabolites, including very long-chain and branched-chain fatty acids in plasma and plasmalogens in red blood cells. Molecular testing can confirm biochemical findings and identify the specific genetic defect, usually utilizing a multiple-gene panel or exome/genome approach. When next-generation sequencing is used as a first-tier test, evaluation of peroxisome metabolism is often necessary to assess the significance of unknown variants and establish the extent of peroxisome dysfunction. This document provides a resource for laboratories developing and implementing clinical biochemical genetic testing for peroxisomal disorders, emphasizing technical considerations for sample collection, test performance, and result interpretation. Additionally, considerations on confirmatory molecular testing are discussed.

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase.

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

ACBD5 deficiency causes a defect in peroxisomal very long-chain fatty acid metabolism.

BACKGROUND: Acyl-CoA binding domain containing protein 5 (ACBD5) is a peroxisomal membrane protein with a cytosolic acyl-CoA binding domain. Because of its acyl-CoA binding domain, ACBD5 has been assumed to function as an intracellular carrier of acyl-CoA esters. In addition, a role for ACBD5 in pexophagy has been suggested. However, the precise role of ACBD5 in peroxisomal metabolism and/or functioning has not yet been established. Previously, a genetic ACBD5 deficiency was identified in three siblings with retinal dystrophy and white matter disease. We identified a pathogenic mutation in ACBD5 in another patient and studied the consequences of the ACBD5 defect in patient material and in ACBD5-deficient HeLa cells to uncover this role. METHODS: We studied a girl who presented with progressive leukodystrophy, syndromic cleft palate, ataxia and retinal dystrophy. We performed biochemical, cell biological and molecular studies in patient material and in ACBD5-deficient HeLa cells generated by CRISPR-Cas9 genome editing. RESULTS: We identified a homozygous deleterious indel mutation in ACBD5, leading to complete loss of ACBD5 protein in the patient. Our studies showed that ACBD5 deficiency leads to accumulation of very long-chain fatty acids (VLCFAs) due to impaired peroxisomal ß-oxidation. No effect on pexophagy was found. CONCLUSIONS: Our investigations strongly suggest that ACBD5 plays an important role in sequestering C26-CoA in the cytosol and thereby facilitates transport into the peroxisome and subsequent ß-oxidation. Accordingly, ACBD5 deficiency is a novel single peroxisomal enzyme deficiency caused by impaired VLCFA metabolism, leading to retinal dystrophy and white matter disease.

Role of substrate unbinding in Michaelis-Menten enzymatic reactions.

The Michaelis-Menten equation provides a hundred-year-old prediction by which any increase in the rate of substrate unbinding will decrease the rate of enzymatic turnover. Surprisingly, this prediction was never tested experimentally nor was it scrutinized using modern theoretical tools. Here we show that unbinding may also speed up enzymatic turnover--turning a spotlight to the fact that its actual role in enzymatic catalysis remains to be determined experimentally. Analytically constructing the unbinding phase space, we identify four distinct categories of unbinding: inhibitory, excitatory, superexcitatory, and restorative. A transition in which the effect of unbinding changes from inhibitory to excitatory as substrate concentrations increase, and an overlooked tradeoff between the speed and efficiency of enzymatic reactions, are naturally unveiled as a result. The theory presented herein motivates, and allows the interpretation of, groundbreaking experiments in which existing single-molecule manipulation techniques will be adapted for the purpose of measuring enzymatic turnover under a controlled variation of unbinding rates. As we hereby show, these experiments will not only shed first light on the role of unbinding but will also allow one to determine the time distribution required for the completion of the catalytic step in isolation from the rest of the enzymatic turnover cycle.

Single-molecule enzymology based on the principle of the Millikan oil drop experiment.

The ability to monitor the progress of single-molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan oil drop experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single-enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions that result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized that allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using dark field microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single-molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties to reduce and/or shield the number of unproductive charges and allow for improved sensitivity.

Towards a Self-Powered Amperometric Glucose Biosensor Based on a Single-Enzyme Biofuel Cell.

This paper describes the study of an amperometric glucose biosensor based on an enzymatic biofuel cell consisting of a bioanode and a biocathode modified with the same enzyme-glucose oxidase (GOx). A graphite rod electrode (GRE) was electrochemically modified with a layer of Prussian blue (PB) nanoparticles embedded in a poly(pyrrole-2-carboxylic acid) (PPCA) shell, and an additional layer of PPCA and was used as the cathode. A GRE modified with a nanocomposite composed of poly(1,10-phenanthroline-5,6-dione) (PPD) and gold nanoparticles (AuNPs) entrapped in a PPCA shell was used as an anode. Both electrodes were modified with GOx by covalently bonding the enzyme to the carboxyl groups of PPCA. The developed biosensor exhibited a wide linear range of 0.15-124.00 mM with an R2 of 0.9998 and a sensitivity of 0.16 µA/mM. The limit of detection (LOD) and quantification (LOQ) were found to be 0.07 and 0.23 mM, respectively. The biosensor demonstrated exceptional selectivity to glucose and operational stability throughout 35 days, as well as good reproducibility, repeatability, and anti-interference ability towards common interfering substances. The studies on human serum demonstrate the ability of the newly designed biosensor to determine glucose in complex real samples at clinically relevant concentrations.

Predicting the Glycemic Index of Biscuits Using Static In Vitro Digestion Protocols.

In vitro digestion methods that can accurately predict the estimated GI (eGI) values of complex carbohydrate foods, including biscuits, are worth exploring. In the current study, standard commercial biscuits with varied clinical GI values between 9~30 were digested using both the INFOGEST and single-enzyme digestion protocols. The digestion kinetic parameters were acquired through mathematical fitting by mathematical kinetics models. The results showed that compared with the INFOGEST protocol, the AUR180 deduced from digesting using either porcine pancreatin or α-amylase showed the best potential in predicting the eGI values. Accordingly, mathematical equations were established based on the relations between the AUR180 and the GI values. When digesting using porcine pancreatin, GI= 1.834 + 0.009 ×AUCR180 (R2= 0.952), and when digesting using only α-amylase, GI= 6.101 + 0.009 ×AUCR180 (R2=0.902). The AUR180 represents the area under the curve of the reducing-sugar content normalized to the total carbohydrates versus the digestion time in 180 min. The in vitro method presented enabled the rapid and accurate prediction of the eGI values of biscuits, and the validity of the formula was verified by another batch of biscuits with a known GI, and the error rate of most samples was less than 30%.

Probing conformational kinetics of catalase with and without magnetic field by single-entity collision electrochemistry.

The conformational motions of enzymes are crucial for their catalytic activities, but these fluctuations are usually spontaneous and unsynchronized and thus difficult to obtain from ensemble-averaged measurements. Here, we employ label-free single-entity electrochemical measurements to monitor in real time the fluctuating enzymatic behavior of single catalase molecules toward the degradation of hydrogen peroxide. By probing the electrochemical signals of single catalase molecules at a carbon nanoelectrode, we were able to observe three distinct current traces that could be attributed to conformational changes on the sub-millisecond timescale. Whereas, nearly uniform single long peaks were observed for single catalase molecules under a moderate magnetic field due to the restricted conformational changes of catalase. By combining high-resolution current signals with a multiphysics simulation model, we studied the catalytic kinetics of catalase with and without a magnetic field, and further estimated the maximum catalytic rate and conformational transition rate. This work introduces a new complementary approach to existing single-molecule enzymology, giving further insight into the enzymatic reaction mechanism.

Recent advances in enzymatic biofuel cells enabled by innovative materials and techniques.

The past few decades have seen increasingly rapid advances in the field of sustainable energy technologies. As a new bio- and eco-friendly energy source, enzymatic biofuel cells (EBFCs) have garnered significant research interest due to their capacity to power implantable bioelectronics, portable devices, and biosensors by utilizing biomass as fuel under mild circumstances. Nonetheless, numerous obstacles impeded the commercialization of EBFCs, including their relatively modest power output and poor long-term stability of enzymes. To depict the current progress of EBFC and address the challenges it faces, this review traces back the evolution of EBFC and focuses on contemporary advances such as newly emerged multi or single enzyme systems, various porous framework-enzyme composites techniques, and innovative applications. Besides emphasizing current achievements in this field, from our perspective part we also introduced novel electrode and cell design for highly effective EBFC fabrication. We believe this review will assist readers in comprehending the basic research and applications of EBFCs as well as potentially spark interdisciplinary collaboration for addressing the pressing issues in this field.

Controlled growth of redox polymer network on single enzyme molecule for stable and sensitive enzyme electrode.

The electrochemical applications of enzymes are often hampered by poor enzyme stability and low electron conductivity. In this work, a novel enzyme nanogel based on atom transfer radical polymerization (ATRP) has been developed for highly sensitive detection of glucose based on ferrocene (Fc) embedded in crosslinked polymer network nanogel. Enzyme surfaces are successively modified with Br initiator, and then in situ atom transfer radical polymerization (ATRP) was performed to build up crosslinked polyacrylamide network. The resulting single enzyme nanogel (ATRP-SEG) is uniform in size fairly. ATRP-SEG reveals bi-phasic inactivation, and the half-life of stable ATRP-SEG after 18-day incubation at 50 °C is 47 days, which is 197 times longer than that of free Gox (5.7 h). By introducing a ferrocene (Fc) containing redox polymer, poly(acrylamide-co-vinylferrocene), the half-life of Fc-ATRP-SEG after 18-day incubation at 50 °C is 49 days. Fc-ATRP-SEG is used for preparation of glucose-sensing electrode, and the sensitivity of Fc-ATRP-SEG electrode is 111 µA cm-2 mM-1, which is 366 and 1270 times higher than those of free GOx (0.303 µA cm-2 mM-1) and ATRP-SEG (0.0874 µA cm-2 mM-1), respectively. Fc-ATRP-SEG electrode maintained 90% of initial current density under 4 °C storage condition and repetitive usages every day for 7 days. Even the electrode repeatedly used in continuous harsh condition (250 rpm, room temperature), the current density maintained 96% after 12 h incubation at operational condition.

Machine Learning on a Robotic Platform for the Design of Polymer-Protein Hybrids.

Polymer-protein hybrids are intriguing materials that can bolster protein stability in non-native environments, thereby enhancing their utility in diverse medicinal, commercial, and industrial applications. One stabilization strategy involves designing synthetic random copolymers with compositions attuned to the protein surface, but rational design is complicated by the vast chemical and composition space. Here, a strategy is reported to design protein-stabilizing copolymers based on active machine learning, facilitated by automated material synthesis and characterization platforms. The versatility and robustness of the approach is demonstrated by the successful identification of copolymers that preserve, or even enhance, the activity of three chemically distinct enzymes following exposure to thermal denaturing conditions. Although systematic screening results in mixed success, active learning appropriately identifies unique and effective copolymer chemistries for the stabilization of each enzyme. Overall, this work broadens the capabilities to design fit-for-purpose synthetic copolymers that promote or otherwise manipulate protein activity, with extensions toward the design of robust polymer-protein hybrid materials.

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay.

Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully-active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, kcat , and the rate constant of productive binding, kon , of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.

Single enzyme nanoparticle, an effective tool for enzyme replacement therapy.

The term "single enzyme nanoparticle" (SEN) refers to a chemically or biologically engineered single enzyme molecule. SENs are distinguished from conventional protein nanoparticles in that they can maintain their individual structure and enzymatic activity following modification. Furthermore, SENs exhibit enhanced properties as biopharmaceuticals, such as reduced antigenicity, and increased stability and targetability, which are attributed to the introduction of specific moieties, such as poly(ethylene glycol), carbohydrates, and antibodies. Enzyme replacement therapy (ERT) is a crucial therapeutic option for controlling enzyme-deficiency-related disorders. However, the unfavorable properties of enzymes, including immunogenicity, lack of targetability, and instability, can undermine the clinical significance of ERT. As shown in the cases of Adagen®, Revcovi®, Palynziq®, and Strensiq®, SEN can be an effective technology for overcoming these obstacles. Based on these four licensed products, we expect that additional SENs will be introduced for ERT in the near future. In this article, we review the concepts and features of SENs, as well as their preparation methods. Additionally, we summarize different types of enzyme deficiency disorders and the corresponding therapeutic enzymes. Finally, we focus on the current status of SENs in ERT by reviewing FDA-approved products.

Tyrosinase nanocapsule based nano-biosensor for ultrasensitive and rapid detection of bisphenol A with excellent stability in different application scenarios.

Bisphenol A (BPA), one of the most important endocrine disrupting chemicals, is a threat to human and wildlife health. Electrochemical enzyme biosensor has been regarded as ideal alternative analytical technique for ultrasensitive and rapid detection of BPA, while the unstable and easily deactivated nature of enzyme limits its development. In order to improve the stability of enzyme, tyrosinase was chosen as a model enzyme, and tyrosinase nanocapsules (nTyr) were prepared by encapsulating a single tyrosinase molecule into a thin network polymer shell through in-situ polymerization method in aqueous solution. The characterization of particle size distribution, TEM and FTIR indicated the successful formation of single tyrosinase molecule nanocapsule. And the porous network polymer shell of nTyr ensured the maintenance of tyrosinase activity and fast substrate transportation. The obtained nTyr was used to construct an electrochemical biosensor for BPA detection, exhibiting a low detection limit of 12 nmol L-1 and a wide linear range from 5 × 10-8 to 2 × 10-6 mol L-1. Compared with native tyrosinase, the nTyr based biosensor displayed dramatically enhanced stability including thermal stability, organic solvent tolerance and acid/base tolerance. The excellent performance of nTyr based biosensor was not only attributed to the protection of biocompatible rigid polymer shells, but also the multipoint covalent attachments between tyrosinase cores and polymer shells. The robust biosensor was further used for rapid detection of BPA leached from plastic products with satisfactory results. The nTyr based nano-biosensor provides a prospective solution to resolve the stability problem of enzyme biosensors in different application scenarios.

The transpeptidase PBP2 governs initial localization and activity of the major cell-wall synthesis machinery in E. coli.

Bacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive 'Rod complex'. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. Using single-particle tracking of the transpeptidase and Rod-complex component PBP2, we found that PBP2 binds to a substrate different from MreB. Depletion and localization experiments of other putative Rod-complex components provide evidence that none of those provide the sole rate-limiting substrate for PBP2 binding. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. We therefore speculate that the local cell-wall architecture provides the cue for Rod-complex initiation, either through direct binding by PBP2 or through an unknown intermediate.

Single enzyme-based stem-loop and linear primers co-mediated exponential amplification of short gene sequences.

Isothermal DNA amplification only using a Bacillus stearothermophilus (Bst) DNA polymerase such as loop-mediated isothermal amplification typically entails multiple target sites for primer design and is thereby not suited for the amplification of short gene sequences, for example, the sequences with size below 200 nucleotides (nt). Here we present SLIMP, a novel single enzyme-based isothermal amplification of short gene sequence mediated by both stem-loop and linear primers. In SLIMP, a pair of stem-loop primers and a pair of linear primers are specifically designed to recognize only two target sites. Linear primers in SLIMP are similar as conventional PCR primers, but stem-loop primers are the modified linear primers through attaching a stem-loop structure at their 5'-ends. Attributed to this unique primer design, three basic reaction modes including linear-priming, single stem-loop-priming, and double stem-loop-priming amplifications co-mediate the SLIMP process under the function of Bst DNA polymerase. As a proof-of-concept assay, a synthetic 80 nt sequence from hepatitis B virus S gene was used as the template to develop SLIMP. On performance, SLIMP detection possesses high sensitivity and specificity, good selectivity, and the potential for analysing real sample. Therefore, SLIMP is expected as a novel alternative to amplify short gene sequences using a single enzyme.

A single enzyme PCR-RFLP protocol targeting 16S rRNA/tRNAval region to authenticate four commercially important shrimp species in India.

Food authenticity is an issue of major concern for food authorities, as mislabeling represents one of the major commercial frauds. In this study, a novel PCR-RFLP protocol was developed as a tool to authenticate four shrimp products of commercial importance belonging to the family, Penaeidae, viz. Litopenaeus vannamei, Penaeus monodon, P. semisulcatus and Fenneropenaeus indicus. PCR amplification was performed targeting 16S rRNA/tRNAval region having an amplicon size of 530bp using the specific primers for shrimps, 16S-Cru4/16S-Cru3. Subsequent restriction analysis with a single restriction enzyme, Tsp5091, yielded distinct RFLP pattern for each species of shrimps having fragment sizes below 150bp. The unique RFLP patterns were also obtained in processed shrimp products without any degradation or alteration in the major fragments. The method was also validated with commercial shrimp products. Thus, the developed protocol can be performed within 8h using a single enzyme to authenticate four shrimp products of commercial significance.

Single Enzyme Direct Biomineralization of CdSe and CdSe-CdS Core-Shell Quantum Dots.

Biomineralization is the process by which biological systems synthesize inorganic materials. Herein, we demonstrate an engineered cystathionine γ-lyase enzyme, smCSE that is active for the direct aqueous phase biomineralization of CdSe and CdSe-CdS core-shell nanocrystals. The nanocrystals are formed in an otherwise unreactive buffered solution of Cd acetate and selenocystine through enzymatic turnover of the selenocystine to form a reactive precursor, likely H2Se. The particle size of the CdSe core nanocrystals can be tuned by varying the incubation time to generated particle sizes between 2.74 ± 0.63 nm and 4.78 ± 1.16 nm formed after 20 min and 24 h of incubation, respectively. Subsequent purification and introduction of l-cysteine as a sulfur source facilitates the biomineralization of a CdS shell onto the CdSe cores. The quantum yield of the resulting CdSe-CdS core-shell particles is up to 12% in the aqueous phase; comparable to that reported for more traditional chemical synthesis routes for core-shell particles of similar size with similar shell coverage. This single-enzyme route to functional nanocrystals synthesis reveals the powerful potential of biomineralization processes.

Label-free optical detection of single enzyme-reactant reactions and associated conformational changes.

Monitoring the kinetics and conformational dynamics of single enzymes is crucial to better understand their biological functions because these motions and structural dynamics are usually unsynchronized among the molecules. However, detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single-molecule basis remains as a challenge to established optical techniques because of the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually nontrivial, and the labels themselves might skew the physical properties of the enzyme. We demonstrate an optical, label-free method capable of observing enzymatic interactions and associated conformational changes on a single-molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we use two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches, we find that low and high polymerase activities can be clearly discerned through their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.

Quantification of Functional Dynamics of Membrane Proteins Reconstituted in Nanodiscs Membranes by Single Turnover Functional Readout.

Single-molecule measurements are emerging as a powerful tool to study the individual behavior of biomolecules, revolutionizing our understanding of biological processes. Their ability to measure the distribution of behaviors, instead of the average behavior, allows the direct observation and quantification of the activity, abundance, and lifetime of multiple states and transient intermediates in the energy landscape that are typically averaged out in nonsynchronized ensemble measurements. Studying the function of membrane proteins at the single-molecule level remains a formidable challenge, and to date there is limited number of available functional assays. In this chapter, we describe in detail our recently developed methodology to reconstitute membrane proteins such as the integral membrane protein cytochrome P450 oxidoreductase on membrane systems such as Nanodiscs and study their functional dynamics by recordings at the fundamental resolution of individual catalytic turnovers using prefluorescent substrate analogues. We initially describe the methodology for reconstitution, surface immobilization, and data acquisition of individual enzyme catalytic turnovers. We then explain in detail the statistical analysis, with an emphasis on the model development, the potential pitfalls for correctly identifying the abundance, lifetime, and likelihood of sampling protein functional states. This methodology may enable studies of functional dynamics and their role in biology for a spectrum of membrane proteins.