Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes.

A novel injection scheme is described in which ultrasmall samples in the attoliter (10(-18) L) and low femtoliter (10(-15) L) range, or even single molecules, are controllably introduced into a tapered capillary so that electrophoretic separation can be carried out. To match the dimensions of the capillary inlet with that of the sample, capillary tips are tapered to an inside diameter ranging from hundreds of nanometers to a few micrometers. To inject an ultrasmall sample, optical trapping is used to immobilize and manipulate the sample in order to place it inside or next to the capillary inlet. A small controlled suction results in the loading of the sample into the capillary.

We have developed a model to study the role of geometrical factors in influencing the early stages of unfolding in three cytochromes: cyt c′, cyt c-b562 and cyt c. Each stage in unfolding is quantified by the spatial extension 〈λ̂i〉 of n-residue segments, and by their angular extension 〈βn〉. Similarities and differences between and among the three cytochromes in the unfolding of helical and non-helical regions can be determined by analyzing the data for each signature separately. Definite conclusions can be drawn when spatial and angular changes are considered in tandem. To facilitate comparisons, we present graphical portraits of the three cytochromes at the same stage of unfolding, and in relation to their native state structures. We also display specific segments at different stages of unfolding to illustrate differences in stability of defined domains thereby allowing us to make specific predictions on the unfolding of corresponding internal and terminal helices in cyt c′ and cyt c-b562. Our work accords with an earlier experimental report on the presence and persistence of a hydrophobic core in cyt c.

We have investigated the structural stability of the SARS (Severe acute respiratory syndrome)-CoV-2 main protease monomer (Mpro). We quantified the spatial and angular changes in the structure using two independent analyses, one based on a spatial metrics (δ, ratio), the second on angular metrics. The order of unfolding of the 10 helices in Mpro is characterized by beta vs alpha plots similar to those of cytochromes and globins. The longest turning region is anomalous in the earliest stage of unfolding. In an investigation of excluded-volume effects, we found that the maximum spread in average molecular-volume values for Mpro, cytochrome c -b 562 , cytochrome c ’, myoglobin, and cytoglobin is ~10 Å 3 . This apparent universality is a consequence of the dominant contributions from six residues: ALA, ASP, GLU, LEU, LYS and VAL. Of the seven Mpro histidines, residues 41, 163, 164, and 246 are in stable H-bonded regions; metal ion binding to one or more of these residues could break up the H-bond network, thereby affecting protease function. Our analysis also indicated that metal binding to cysteine residues 44 and 145 could disable the enzyme.

We study the interplay between entropic and energetic factors in influencing the efficiency with which linear r-mers sieve through a cavity when the size of the r-mer is commensurate with the spatial dimensions of the system. We consider here monomers, dimers, and linear trimers and determine the mean transit time 7 (related to the mean walk length ( n ) ) for the r-mer, entering thecavity at one end, to exit at the other. We study first r-mer diffusion in twodimensiolls and then systematically expand the diffusion space in our (lattice) model by adding layers in the third dimension. To examine the effect of an increase in temperature or a decrease in viscoSity of the carrier medium, diffusion involving strictly translational modes is contrasted with motion of the diffusing r-mer in which both translation and pivoting (and hence "tumbling") of the r-mer is permitted. Although one anticipates that an unstructured (monomeric) species would sieve through a constrained space more rapidly than a more structured dimer or trimer, our calculations show that this behavior is not universal; structured r-mers have, by virtue of that structure, accc88 to a smaller "excluded volume" (Le., a smaller fraction of the available phase space) internal to the cavity and hence their passage through the cavity can be more facile than an unstructured monomer. However, if monomers, dimers, and trimers confront "activation barriers" in their sitatmite migration through the system, our calculations show that energetic effects soon dominate entropic ones, and the sieving efficiency decreases systematically with increase in the chain length of the r-mer. The trends observed are compared with those reported in the experimental study of Caro et al. (J. Chem. Soc., Faraday Tram. I 1985,81, 2541) and the recent molecular dynamics simulations of Nowak et al. (J. Phys. Chem. 1991, 95, 848) on the diffusivity of methane, ethane, and propane in silicalite. IntrOdllCtiOllRecently, Nowak et al.' reported molecular dynamics simulations performed to study the influence of zeolite structure (specihlly, the all-silica polymorphs of zeolite EU-1. mordenite, and silicalite) on the migration of methane in the zeolite void s p a .They also reported diffusion simulations of the structured aliphatic molecules ethane and propane in silicalite and compared the results obtained with those previously reported in the experimental study of Caro et alqz A brief recapitulation of their results follows. Nowak et al.' found that an increme in pore diameter of the zeolite mimhannel led to an increase in the migration rate of methane. For structured molecules, their computed diffusion characteristics were in qualitative agreement with the trends found by Caro et namely, that the diffusion caffcient decreased with increase in the carbon chain length. Quantitatively, their predicted diffusivities were in good agreement with the experimental results of Car0 et al? but less good for propane. Their results for propane were attributed "to a computational artifact stemming from ...