Activity Of Individual Molecules Research Articles

Can Protein Conformers Be Fractionated by Crystallization?

Molecular crystallization typically singles out a specific conformation, or a set of conformations that are identical over large parts and may show some flexibility, from a mixture of equilibrating conformations in solution. To critically evaluate the selectivity of this process, human lactate dehydrogenase isozyme 1 (LDH-1) microcrystals were separately dissolved and subsequently assayed inside capillaries with electrophoretically mediated microanalysis (EMMA) at both the ensemble and the single-molecule level. While fragments from the same crystal exhibited identical enzyme activities, different crystals, even when grown from the same drop of mother liquor, showed markedly different activities. Activities of individual molecules from a crystal were found to be essentially identical, whereas molecules obtained directly from solution showed a 4-fold variation in activity. Furthermore, after storage at 37 °C, the distribution of single-molecule LDH activities from solutions of individual crystals broadened and approached that of LDH obtained from the original solution. X-ray crystallography also showed distinct conformations for single microcrystals and confirms that crystallization properly selects even small conformational variants of proteins and that the slow equilibration to multiple stable conformations in solution is responsible for the observed single-molecule heterogeneity.

A simple system for the measurement of the distribution of activities of individual molecules of E. coli β-galactosidase

A simple system capable of measuring the distribution of single molecule activities of a population of E. coli β-galactosidase was developed. This system utilized a modified disposable syringe, a narrow bore capillary tube and an in-laboratory constructed laser-induced fluorescence detection system. Once started, the experiment can continue to collect data without any operator input for more than 24 h. The relative catalytic rates of 138 individual molecules of the enzyme were determined. The distribution of activities was found to be indistinguishable from that reported using more complicated methodologies.

Single Molecule Detection of One, Two and Multiplex Proteins Involved in DNA/RNA Transaction

Cellular processes involve complex arrangement of proteins engaged in a multitude of reactions, yet in a highly coordinated manner. The level of complexity, however, makes it difficult to investigate the role played by the individual protein constituent. Data taken from the conventional bulk solution methods suffer from ensemble averaging effect in which information from individual molecules is masked. The single molecule detection method overcomes this limitation by offering unique tools for monitoring the activity of individual molecules in isolation and in real-time dynamics. Included in this review are recent articles of single molecule studies representing a diverse array of experimental platforms which demonstrate the power and spectrum of single molecule detection.

TABASCO: A single molecule, base-pair resolved gene expression simulator.

BackgroundExperimental studies of gene expression have identified some of the individual molecular components and elementary reactions that comprise and control cellular behavior. Given our current understanding of gene expression, and the goals of biotechnology research, both scientists and engineers would benefit from detailed simulators that can explicitly compute genome-wide expression levels as a function of individual molecular events, including the activities and interactions of molecules on DNA at single base pair resolution. However, for practical reasons including computational tractability, available simulators have not been able to represent genome-scale models of gene expression at this level of detail.ResultsHere we develop a simulator, TABASCO , which enables the precise representation of individual molecules and events in gene expression for genome-scale systems. We use a single molecule computational engine to track individual molecules interacting with and along nucleic acid polymers at single base resolution. Tabasco uses logical rules to automatically update and delimit the set of species and reactions that comprise a system during simulation, thereby avoiding the need for a priori specification of all possible combinations of molecules and reaction events. We confirm that single molecule, base-pair resolved simulation using TABASCO (Tabasco) can accurately compute gene expression dynamics and, moving beyond previous simulators, provide for the direct representation of intermolecular events such as polymerase collisions and promoter occlusion. We demonstrate the computational capacity of Tabasco by simulating the entirety of gene expression during bacteriophage T7 infection; for reference, the 39,937 base pair T7 genome encodes 56 genes that are transcribed by two types of RNA polymerases active across 22 promoters.ConclusionTabasco enables genome-scale simulation of transcription and translation at individual molecule and single base-pair resolution. By directly representing the position and activity of individual molecules on DNA, Tabasco can directly test the effects of detailed molecular processes on system-wide gene expression. Tabasco would also be useful for studying the complex regulatory mechanisms controlling eukaryotic gene expression. The computational engine underlying Tabasco could also be adapted to represent other types of processive systems in which individual reaction events are organized across a single spatial dimension (e.g., polysaccharide synthesis).

Single Molecules of Highly Purified Bacterial Alkaline Phosphatase Have Identical Activity

The central paradigm of chemistry is that molecular structure determines molecular function. Details of this paradigm can be tested with single-molecule enzymology, where the activity of individual molecules is studied. In all cases reported thus far, there is a large molecule-to-molecule heterogeneity in activity and activation energy. This heterogeneity must arise from differences in structure. Replicate incubations on the same molecule yield consistent results; the structural heterogeneity must be stable over the time period of the experiment, which can extend over several hours. In this paper, we demonstrate that highly purified molecules of bacterial alkaline phosphatase generate identical activity; structurally identical molecules behave identically. In contrast, the glycosylated mammalian enzyme demonstrates a complex isoelectric focusing pattern and has a dramatic molecule-to-molecule variation in activity and activation energy. Glycosylation affects both the kinetics and energetics of this enzymatically catalyzed reaction.

<i>Escherichia coli</i> β-galactosidase is heterogeneous with respect to the activity of individual molecules

<i>Escherichia coli</i> β-galactosidase is heterogeneous with respect to the activity of individual molecules