Conditional ambiguity of one‐dimensional crystal structures determined from a minimum of diffraction intensity data

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113118/1/S0108767311007616.pd

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

Contributions of Coulombic and Hofmeister Effects to the Osmotic Activation of <i>Escherichia coli</i> Transporter ProP

Osmosensing transporters mediate osmolyte accumulation to forestall cellular dehydration as the extracellular osmolality increases. ProP is a bacterial osmolyte-H⁺ symporter, a major facilitator superfamily member, and a paradigm for osmosensing. ProP activity is a sigmoid function of the osmolality. It is determined by the osmolality, not the magnitude or direction of the osmotic shift, in cells and salt-loaded proteoliposomes. The activation threshold varies directly with the proportion of anionic phospholipid in cells and proteoliposomes. The osmosensory mechanism was probed by varying the salt composition and concentration outside and inside proteoliposomes. Data analysis was based on the hypothesis that the fraction of maximal transporter activity at a particular luminal salt concentration reflects the proportion of ProP molecules in an active conformation. ProP attained the same activity at the same osmolality when diverse, membrane-impermeant salts were added to the external medium. Contributions of Coulombic and/or Hofmeister salt effects to ProP activation were examined by varying the luminal salt cation (K⁺ and Na⁺) and anion (chloride, phosphate, and sulfate) composition and then systematically increasing the luminal salt concentration by increasing the external osmolality. ProP activity increased with the sixth power of the univalent cation concentration, independent of the type of anion. This indicates that salt activation of ProP is a Coulombic, cation effect resulting from salt cation accumulation and not site-specific cation binding. Possible origins of this Coulombic effect include folding or assembly of anionic cytoplasmic ProP domains, an increase in local membrane surface charge density, and/or the juxtaposition of anionic protein and membrane surfaces during activation

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 (〈φ〉) 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 〈φ〉 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 (DGFP) in the cytoplasm of E. coli cells as a function of 〈φ〉 for both plasmolyzed and adapted cells. For plasmolyzed cells, the median DGFP (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}D_{GFP}^{m}\end{equation*}\end{document}) decreases by a factor of 70 as 〈φ〉 increases from 0.16 to 0.33. In sharp contrast, for adapted cells, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}D_{GFP}^{m}\end{equation*}\end{document} decreases only by a factor of 2.1 as 〈φ〉 increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by 〈φ〉 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

Quantifying Interactions of Nucleobase Atoms with Model Compounds for the Peptide Backbone and Glutamine and Asparagine Side Chains in Water

Alkylureas display hydrocarbon and amide groups, the primary functional groups of proteins. To obtain the thermodynamic information that is needed to analyze interactions of amides and proteins with nucleobases and nucleic acids, we quantify preferential interactions of alkylureas with nucleobases differing in the amount and composition of water-accessible surface area (ASA) by solubility assays. Using an established additive ASA-based analysis, we interpret these thermodynamic results to determine interactions of each alkylurea with five types of nucleobase unified atoms (carbonyl sp²O, amino sp³N, ring sp²N, methyl sp³C, and ring sp²C). All alkylureas interact favorably with nucleobase sp²C and sp³C atoms; these interactions become more favorable with an increasing level of alkylation of urea. Interactions with nucleobase sp²O are most favorable for urea, less favorable for methylurea and ethylurea, and unfavorable for dialkylated ureas. Contributions to overall alkylurea–nucleobase interactions from interactions with each nucleobase atom type are proportional to the ASA of that atom type with proportionality constant (interaction strength) α, as observed previously for urea. Trends in α-values for interactions of alkylureas with nucleobase atom types parallel those for corresponding amide compound atom types, offset because nucleobase α-values are more favorable. Comparisons between ethylated and methylated ureas show interactions of amide compound sp³C with nucleobase sp²C, sp³C, sp²N, and sp³N atoms are favorable while amide sp³C–nucleobase sp²O interactions are unfavorable. Strongly favorable interactions of urea with nucleobase sp²O but weakly favorable interactions with nucleobase sp³N indicate that amide sp²N–nucleobase sp²O and nucleobase sp³N–amide sp²O hydrogen bonding (NH···OC) interactions are favorable while amide sp²N–nucleobase sp³N interactions are unfavorable. These favorable amide–nucleobase hydrogen bonding interactions are prevalent in specific protein–nucleotide complexes

Experimental Atom-by-Atom Dissection of Amide–Amide and Amide–Hydrocarbon Interactions in H₂O

Quantitative information about amide interactions in water is needed to understand their contributions to protein folding and amide effects on aqueous processes and to compare with computer simulations. Here we quantify interactions of urea, alkylated ureas, and other amides by osmometry and amide–aromatic hydrocarbon interactions by solubility. Analysis of these data yields strengths of interaction of ureas and naphthalene with amide sp²O, amide sp²N, aliphatic sp³C, and amide and aromatic sp²C unified atoms in water. Interactions of amide sp²O with urea and naphthalene are favorable, while amide sp²O–alkylurea interactions are unfavorable, becoming more unfavorable with increasing alkylation. Hence, amide sp²O–amide sp²N interactions (proposed n−σ* hydrogen bond) and amide sp²O–aromatic sp²C (proposed n−π*) interactions are favorable in water, while amide sp²O–sp³C interactions are unfavorable. Interactions of all ureas with sp³C and amide sp²N are favorable and increase in strength with increasing alkylation, indicating favorable sp³C–amide sp²N and sp³C–sp³C interactions. Naphthalene results show that aromatic sp²C–amide sp²N interactions in water are unfavorable while sp²C–sp³C interactions are favorable. These results allow interactions of amide and hydrocarbon moieties and effects of urea and alkylureas on aqueous processes to be predicted or interpreted in terms of structural information. We predict strengths of favorable urea–benzene and <i>N</i>-methylacetamide interactions from experimental information to compare with simulations and indicate how amounts of hydrocarbon and amide surfaces buried in protein folding and other biopolymer processes and transition states can be determined from analysis of urea and diethylurea effects on equilibrium and rate constants