Light scattering studies on Ficoll PM70 solutions reveal two distinct diffusive modes

Georgalis, Yannis; Philipp, Martine; Aleksandrova, Ralitsa; Krüger, Jan

doi:10.1016/j.jcis.2012.07.062

Cited by 22 publications

(13 citation statements)

References 26 publications

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“…Separations in Ficoll normally result in better preservation of cell function and morphology. We used different concentrations of Ficoll (1-10%) to planktonic cells (Georgalis et al 2012). FCCP, carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone is a widely used protonophore responsible for the collapse of the chemiosmotic membrane potential (Armitage 2001;Kelly and Thomas 2001).…”

Section: Methodsmentioning

confidence: 99%

Stoichiometry and kinetics of mercury uptake by photosynthetic bacteria

2017

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Mercury adsorption on the cell surface and intracellular uptake by bacteria represent the key first step in the production and accumulation of highly toxic mercury in living organisms. In this work, the biophysical characteristics of the mercury bioaccumulation are studied in intact cells of photosynthetic bacteria by use of analytical (dithizone) assay and physiological photosynthetic markers (pigment content, fluorescence induction and membrane potential) to determine the amount of mercury ions bound to the cell surface and taken up by the cell. It is shown that the Hg(II) uptake mechanism 1) has two kinetically distinguishable components, 2) includes co-opted influx through heavy metal transporters since the slow component is inhibited by Ca 2+ channel blockers, 3) shows complex pH-dependence demonstrating the competition of ligand binding of Hg(II) ions with H + ions (low pH) and high tendency of complex formation of Hg(II) with hydroxyl ions (high pH) and 4) is not a passive but an energy-dependent process as evidenced by light-activation and inhibition by protonophore. Photosynthetic bacteria can accumulate Hg(II) in amounts much (about 10 5 ) greater than their own masses by well defined strong and weak binding sites with equilibrium binding constants in the range of 1 (μM) -1 and 1 (mM) -1 , respectively. The strong binding sites are attributed to sulfhydryl groups as the uptake is blocked by use of sulfhydryl modifying agents and their number is much (two orders of magnitude) smaller than the number of weak binding sites. Biofilms developed by some bacteria (e.g. Rvx. gelatinosus) increase the mercury binding capacity further by a factor of about five. Photosynthetic bacteria in the light act as sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury contaminated aqueous cultures.2

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Section: Methodsmentioning

confidence: 99%

Stoichiometry and kinetics of mercury uptake by photosynthetic bacteria

2017

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“…Using Eq. and a reported hydrodynamic radius

R_{g} = 4.7 nm

, we estimate

c *

for Ficoll to be 270 g L −1 . From that value, we surmise that 300 g L −1 represents a Ficoll concentration that is near the critical overlap concentration

true(bold-italicc \approx bold-italicc *

) and that 400 g L −1 represents a semi‐dilute solution (

bold-italicc > bold-italicc * true)

.…”

Section: Discussionmentioning

confidence: 77%

“…This result makes sense because we expect that weaker helices would be more prone to destabilization by Ficoll. At 300 g L 21 , Ficoll destabilized the N-terminal edge of helical region 1 (residues [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]. Figure 3 shows that peptide 23-32 is the only segment associated with a-1 that was destabilized in the presence of Ficoll.…”

Section: Resultsmentioning

confidence: 99%

“…Ficoll 70, is highly crosslinked and is better described as a semi‐rigid sphere (or star polymer) than as a linear polymer . Based on the molecular weight, approximately 200 sucrose monomers are contained in each Ficoll molecule; all within a radius of only 4.7 nm . Based on these specifications, Ficoll molecules exist in solution as self‐contained spheroidal polymer meshes whose interior resembles a semi‐dilute solution of short linear polymers, as depicted in Figure .…”

Section: Discussionmentioning

confidence: 99%

“…However, Ficoll is not a rigid sphere; molecular sieve experiments suggest that Ficoll is much more flexible in solution than globular proteins of similar Stoke's radius . In addition to being deformable, Ficoll comprises a complex distribution of conformations and molecular weights in solution. Owing to this flexibility and polydispersity, we expect Ficoll to arrange itself differently in 300 g L −1 and 400 g L −1 solutions.…”

Section: Discussionmentioning

confidence: 99%

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Soft interactions and volume exclusion by polymeric crowders can stabilize or destabilize transient structure in disordered proteins depending on polymer concentration

Rusinga

Weis

2017

Proteins

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The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well-understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L and 400 g L of Ficoll, a synthetic polysaccharide, using a recently-developed strong cation exchange-based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275-1282]. Transiently helical regions of ACTR exchanged faster in 300 g L Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L Ficoll is a semi-dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468-1479. © 2017 Wiley Periodicals, Inc.

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