Irina A. Shkel

Diffusion dependent cell behavior in microenvironments.

Understanding the interaction between soluble factors and cells in the cellular microenvironment is critical to understanding a wide range of diseases. Microchannel culture systems provide a tool for separating diffusion and convection based transport making possible controlled studies of the effects of soluble factors in the cellular microenvironment. In this paper we compare the proliferation kinetics of cells in traditional culture flasks to those in microfluidic channels, and explore the relationship between microchannel geometry and cell proliferation. PDMS (polydimethylsiloxane) microfluidic channels were fabricated using micromolding methods. Fall armyworm ovarian cells (Sf9) were homogeneously seeded in a series of different sized microchannels and cultured under a no flow condition. The proliferation rates of Sf9 cells in all of the microchannels were slower than in the flask culture over the first 24 h of culture. The proliferation rates in the microchannels then continuously decreased reaching 5% of that in the flasks over the next 48 h and maintained this level for 5 days. This growth inhibition was reversible and influenced only by the cell seeding density and the channel height but not the channel length or width. One possible explanation for the observed dimension-dependent cell proliferation is the accumulation of different functional molecules in the diffusion dominant microchannel environment. This study provides insights into the potential effects of the diffusion of soluble factors and related effects on cell behavior in microenvironments relevant to the emerging use of microchannel culture systems.

Crowding and Confinement Effects on Protein Diffusion In Vivo

The first in vivo measurements of a protein diffusion coefficient versus cytoplasmic biopolymer volume fraction are presented. Fluorescence recovery after photobleaching yields the effective diffusion coefficient on a 1-mum-length scale of green fluorescent protein within the cytoplasm of Escherichia coli grown in rich medium. Resuspension into hyperosmotic buffer lacking K+ and nutrients extracts cytoplasmic water, systematically increasing mean biopolymer volume fraction, , and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or Delta, of 0), the mean diffusion coefficient, , in cytoplasm (6.1 +/- 2.4 microm2 s(-1)) is only 0.07 of the in vitro value (87 microm2 s(-1)); the relative dispersion among cells, sigmaD/ (standard deviation, sigma(D), relative to the mean), is 0.39. Both and sigmaD/ remain remarkably constant over the range of Delta values of 0 to 0.28 osmolal. For a Delta value of > or =0.28 osmolal, formation of visible plasmolysis spaces (VPSs) coincides with the onset of a rapid decrease in by a factor of 380 over the range of Delta values of 0.28 to 0.70 osmolal and a substantial increase in sigmaD/. Individual values of D vary by a factor of 9 x 10(4) but correlate well with f(VPS), the fractional change in cytoplasmic volume on VPS formation. The analysis reveals two levels of dispersion in D among cells: moderate dispersion at low Delta values for cells lacking a VPS, perhaps related to variation in phi or biopolymer organization during the cell cycle, and stronger dispersion at high Delta values related to variation in f(VPS). Crowding effects alone cannot explain the data, nor do these data alone distinguish crowding from possible binding or confinement effects within a cytoplasmic meshwork.

Separation of preferential interaction and excluded volume effects on DNA duplex and hairpin stability

Small solutes affect protein and nucleic acid processes because of favorable or unfavorable chemical interactions of the solute with the biopolymer surface exposed or buried in the process. Large solutes also exclude volume and affect processes where biopolymer molecularity and/or shape changes. Here, we develop an analysis to separate and interpret or predict excluded volume and chemical effects of a flexible coil polymer on a process. We report a study of the concentration-dependent effects of the full series from monomeric to polymeric PEG on intramolecular hairpin and intermolecular duplex formation by 12-nucleotide DNA strands. We find that chemical effects of PEG on these processes increase in proportion to the product of the amount of DNA surface exposed on melting and the amount of PEG surface that is accessible to this DNA, and these effects are completely described by two interaction terms that quantify the interactions between this DNA surface and PEG end and interior groups. We find that excluded volume effects, once separated from these chemical effects, are quantitatively described by the analytical theory of Hermans, which predicts the excluded volume between a flexible polymer and a rigid molecule. From this analysis, we show that at constant concentration of PEG monomer, increasing PEG size increases the excluded volume effect but decreases the chemical interaction effect, because in a large PEG coil a smaller fraction of the monomers are accessible to the DNA. Volume exclusion by PEG has a much larger effect on intermolecular duplex formation than on intramolecular hairpin formation.

Why Hofmeister effects of many salts favor protein folding but not DNA helix formation

The majority (∼70%) of surface buried in protein folding is hydrocarbon, whereas in DNA helix formation, the majority (∼65%) of surface buried is relatively polar nitrogen and oxygen. Our previous quantification of salt exclusion from hydrocarbon (C) accessible surface area (ASA) and accumulation at amide nitrogen (N) and oxygen (O) ASA leads to a prediction of very different Hofmeister effects on processes that bury mostly polar (N, O) surface compared to the range of effects commonly observed for processes that bury mainly nonpolar (C) surface, e.g., micelle formation and protein folding. Here we quantify the effects of salts on folding of the monomeric DNA binding domain (DBD) of lac repressor (lac DBD) and on formation of an oligomeric DNA duplex. In accord with this prediction, no salt investigated has a stabilizing Hofmeister effect on DNA helix formation. Our ASA-based analyses of model compound data and estimates of the surface area buried in protein folding and DNA helix formation allow us to predict Hofmeister effects on these processes. We observe semiquantitative to quantitative agreement between these predictions and the experimental values, obtained from a novel separation of coulombic and Hofmeister effects. Possible explanations of deviations, including salt-dependent unfolded ensembles and interactions with other types of surface, are discussed.

Cytoplasmic Protein Mobility in Osmotically Stressed Escherichia coli

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.

Protein Diffusion in the Periplasm of E. coli under Osmotic Stress

The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure diffusion of periplasmic green fluorescent protein and probe the fluidity of the periplasm as a function of external osmotic conditions. For cells adapted to growth in complete medium at 0.14-1.02 Osm, the mean diffusion coefficient increases from 3.4 μm² s⁻¹ to 6.6 μm² s⁻¹ and the distribution of D(peri) broadens as growth osmolality increases. This is consistent with a net gain of water by the periplasm, decreasing its biopolymer volume fraction. This supports a model in which the turgor pressure drops primarily across the thin peptidoglycan layer while the cell actively maintains osmotic balance between periplasm and cytoplasm, thus avoiding a substantial pressure differential across the cytoplasmic membrane. After sudden hyperosmotic shock (plasmolysis), the cytoplasm loses water as the periplasm gains water. Accordingly, increases threefold. The fluorescence recovery after photobleaching is complete and homogeneous in all cases, but in minimal medium, the periplasm is evidently thicker at the cell tips. For the relevant geometries, Brownian dynamics simulations in model cytoplasmic and periplasmic volumes provide analytical formulae for extraction of accurate diffusion coefficients from readily measurable quantities.

Chemical Interactions of Polyethylene Glycols (PEGs) and Glycerol with Protein Functional Groups: Applications to Effects of PEG and Glycerol on Protein Processes.

In this work, we obtain the data needed to predict chemical interactions of polyethylene glycols (PEGs) and glycerol with proteins and related organic compounds and thereby interpret or predict chemical effects of PEGs on protein processes. To accomplish this, we determine interactions of glycerol and tetraEG with >30 model compounds displaying the major C, N, and O functional groups of proteins. Analysis of these data yields coefficients (α values) that quantify interactions of glycerol, tetraEG, and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with aromatic C, amide N, and cationic N, but unfavorably with amide O, carboxylate O, and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliphatic C are quite favorable, while interactions of glycerol and PEG end groups with aliphatic C are not. Hence, tetraEG and PEG300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD), while glycerol and di- and monoethylene glycol are stabilizers. Favorable interactions with aromatic and aliphatic C explain why PEG400 greatly increases the solubility of aromatic hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α values reported here, chemical contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA) and separated from excluded volume effects.

Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

Significance Analysis of effects of denaturants and temperature on folding and unfolding rate constants of 13 globular proteins yields the amount and composition of the surface buried in folding to and from the high–free-energy transition state (TS), and thereby provides information about folding mechanisms and early unstable folding intermediates. All 13 proteins preferentially bury amide surface in folding to TS; amounts of amide and hydrocarbon surface buried in folding to TS generally exceed those buried in forming the native secondary structure. From this, we conclude that most native secondary structure forms in early unstable folding intermediates and that conversion of these intermediates to TS involves nucleation of tertiary interactions, allowing preferential burial of hydrocarbon surface as TS folds. Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high–free-energy transition state (TS) will help answer some of these. Here, we use effects of denaturants (urea, guanidinium chloride) and temperature on folding and unfolding rate constants and the overall equilibrium constant as probes of surface area changes in protein folding. We interpret denaturant kinetic m-values and activation heat capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding. Predicted accessible surface area changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50–90% of overall surface burial) and that the surface buried is disproportionately amide, demonstrating extensive formation of secondary structure in early intermediates. Models of possible pre-TS intermediates with all elements of the native secondary structure, created for several of these proteins, bury less amide and hydrocarbon surface than predicted for TS. Therefore, we propose that TS generally has both the native secondary structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.

Separating chemical and excluded volume interactions of polyethylene glycols with native proteins: Comparison with PEG effects on DNA helix formation

Small and large PEGs greatly increase chemical potentials of globular proteins (μ2), thereby favoring precipitation, crystallization, and protein–protein interactions that reduce water‐accessible protein surface and/or protein‐PEG excluded volume. To determine individual contributions of PEG‐protein chemical and excluded volume interactions to μ2 as functions of PEG molality m3, we analyze published chemical potential increments μ23 = dμ2/dm3 quantifying unfavorable interactions of PEG (PEG200‐PEG6000) with BSA and lysozyme. For both proteins, μ23 increases approximately linearly with the number of PEG residues (N3). A 1 molal increase in concentration of PEG ‐CH2OCH2‐ groups, for any chain‐length PEG, increases μBSA by ∼2.7 kcal/mol and μlysozyme by ∼1.0 kcal/mol. These values are similar to predicted chemical interactions of PEG ‐CH2OCH2‐ groups with these protein components (BSA ∼3.3 kcal/mol, lysozyme ∼0.7 kcal/mol), dominated by unfavorable interactions with amide and carboxylate oxygens and counterions. While these chemical effects should be dominant for small PEGs, larger PEGS are expected to exhibit unfavorable excluded volume interactions and reduced chemical interactions because of shielding of PEG residues in PEG flexible coils. We deduce that these excluded volume and chemical shielding contributions largely compensate, explaining why the dependence of μ23 on N3 is similar for both small and large PEGs.

Nonspecific DNA binding and bending by HUαβ: interfaces of the three binding modes characterized by salt-dependent thermodynamics.

Previous isothermal titration calorimetry (ITC) and Förster resonance energy transfer studies demonstrated that Escherichia coli HU(αβ) binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34-bp mode that interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it by 140(o) and thereby increasing its flexibility, and two weaker, modestly cooperative small site-size modes (10 bp and 6 bp) that are useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and we deduce that DNA in the 34-bp mode is bent around-but not wrapped on-the body of HU, in contrast to specific binding of integration host factor. Analyses of binding isotherms (8-bp, 15-bp, and 34-bp DNA) and initial binding heats (34-bp, 38-bp, and 160-bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Sk(i)) even though their binding site sizes differ greatly; the most probable values of Sk(i) on 34-bp DNA or larger DNA are -7.5±0.5. From the similarity of Sk(i) values, we conclude that the binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent DNA 34-bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6-bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Sk(i) values are proposed.