Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.

https://science.sciencemag.org/content/sci/283/5408/1676.full.pdf

Fluorescence spectroscopy of single biomolecules.

Enzymatic turnovers of single cholesterol oxidase molecules were observed in real time by monitoring the emission from the enzyme's fluorescent active site, flavin adenine dinucleotide (FAD). Statistical analyses of single-molecule trajectories revealed a significant and slow fluctuation in the rate of cholesterol oxidation by FAD. The static disorder and dynamic disorder of reaction rates, which are essentially indistinguishable in ensemble-averaged experiments, were determined separately by the real-time single-molecule approach. A molecular memory phenomenon, in which an enzymatic turnover was not independent of its previous turnovers because of a slow fluctuation of protein conformation, was evidenced by spontaneous spectral fluctuation of FAD.

https://science.sciencemag.org/content/sci/282/5395/1877.full.pdf

Single-Molecule Enzymatic Dynamics

The biological and physical sciences share a common interest in small structures (the definition of 'small' depends on the application, but can range from 1 nm to 1 mm). A vigorous trade across the borders of these areas of science is developing around new materials and tools (largely from the physical sciences) and new phenomena (largely from the biological sciences). The physical sciences offer tools for synthesis and fabrication of devices for measuring the characteristics of cells and sub-cellular components, and of materials useful in cell and molecular biology; biology offers a window into the most sophisticated collection of functional nanostructures that exists.

The 'right' size in nanobiotechnology.

By far the largest proportion of the Earth's biosphere is comprised of organisms that thrive in cold environments (psychrophiles). Their ability to proliferate in the cold is predicated on a capacity to synthesize cold-adapted enzymes. These enzymes have evolved a range of structural features that confer a high level of flexibility compared to thermostable homologs. High flexibility, particularly around the active site, is translated into low-activation enthalpy, low-substrate affinity, and high specific activity at low temperatures. High flexibility is also accompanied by a trade-off in stability, resulting in heat lability and, in the few cases studied, cold lability. This review addresses the structure, function, and stability of cold-adapted enzymes, highlighting the challenges for immediate and future consideration. Because of the unique properties of cold-adapted enzymes, they are not only an important focus in extremophile biology, but also represent a valuable model for fundamental researc...

Cold-adapted enzymes.

Recent developments in optical studies of single molecules at room temperature are reviewed, with an emphasis on the underlying principles and the potential of single-molecule experiments. Examples of single-molecule studies are given, including photophysics and photochemistry pertinent to single-molecule measurements, spectral fluctuations, Raman spectroscopy, diffusional motions, conformational dynamics, fluorescence resonant energy transfer, exciton dynamics, and enzymatic turnovers. These studies illustrate the information obtainable with the single-molecule approach that is hidden in ensemble-averaged measurements.

Optical studies of single molecules at room temperature

Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; micr...

Studies on single alkaline phosphatase molecules : reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation : the death of an enzyme

Fluorescent dyes are often used to label proteins before analysis by capillary electrophoresis. Fluorescent labeling produces spectacular improvements in sensitivity compared with UV absorbance detection of the native protein. However, labeling of the protein can lead to significant band broadening. This band broadening is interpreted as a result of multiple labeling of the protein, wherein one or more fluorescent molecules are bound to the protein. The heterogeneous reaction products, which are presumed to have different mobilities, generate a broad peak during electrophoresis. There has been little direct evidence for multiple labeling as the cause of band broadening of proteins. In this paper, we perform electrophoresis on native green fluorescence protein, along with the reaction products produced by fluorescence labeling. For short incubations, a series of regularly spaced components are resolved by free-zone electrophoresis; upon longer incubation, the product peaks merge together, forming a broad e...

Multiple labeling of proteins.

Alkaline phosphatase can be assayed by monitoring the conversion of the fluorogenic substrate AttoPhos into the highly fluorescent product AttoFluor. We have used capillary electrophoresis with laser-induced fluorescence detection to monitor this reaction. The concentration limit of detection (3σ) of alkaline phosphatase is 1.5 × 10-17 M (2.1 fg/mL), which corresponds to a mass limit of detection of nine molecules (1.5 × 10-23 mol) contained within a 1-μL sample volume.

Detection of Attomolar Concentrations of Alkaline Phosphatase by Capillary Electrophoresis Using Laser-Induced Fluorescence Detection

We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser-induced fluorescence Different dyes are used to label standard and sample proteins A two-spectral channel detector resolves fluorescence from the sample and standards Comparison of the migration time of the sample and standards permits the precise determination of molecular weight, irrespective of variations in run-to-run migration times

Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection

Aequorea victoria green fluorescent protein was assayed by capillary electrophoresis using post-capillary laser-induced fluorescence detection in a sheath flow cuvette. The limit of detection was 3.0×10−12 m protein in an injection volume of 17 nL, corresponding to a mass of 3100 molecules. © 1997 John Wiley & Sons, Ltd.

Douglas B. Craig

Papers

Studies on single alkaline phosphatase molecules : reaction rate and activation energy of a reaction catalyzed by a single molecule and the effect of thermal denaturation : the death of an enzyme

Multiple labeling of proteins.

Detection of Attomolar Concentrations of Alkaline Phosphatase by Capillary Electrophoresis Using Laser-Induced Fluorescence Detection

Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection

Detection of Aequorea victoria Green Fluorescent Protein by Capillary Electrophoresis Laser Induced Fluorescence Detection