Laird, Bobby W.

Reinhart, Gregory D.

Elsevier - Publisher Connector 

Monday, February 22, 2010 245a1275-Pos
Characterization of Selectin-Mediated Cell Binding in Shear Flow Using
Micropatterning Technology and Modeling
Konstantinos Konstantopolous, ZiQiu Tong, Luthur Siu-Lun Cheung.
Johns Hopkins University, Baltimore, MD, USA.
Cellular interactions play an essential role in diverse (patho)physiological pro-
cesses such as leukocytes extravasation and cancer metastasis. Selectins initiate
the tethering and rolling of free-flowing cells on activated endothelium before
the integrin-dependent firm adhesion under flow. Utilizing microfluidics de-
vices and protein micropatterning technology, the adhesion kinetics of the
HL-60 leukocyte-like cells to immobilized P-/L-selectin was investigated. Mul-
tiple selectin patches of varying lengths (6-160mm; in the direction of flow) and
constant width (10mm) were patterned on a glass substrate to provide specific
adhesion. A PDMS layer, which had been cured and peeled off from a 25mm
height microchannel mold, was bonded on the patterned glass substrate to
form the microfluidics device. Cell suspensions were perfused through the de-
vice under prescribed shear stresses varying from 0.25-2dyn/cm2. Our data re-
veal the existence of a critical patch length, Lc, which represents the cell rolling
distance to form an initial binding and is a function of the wall shear stress and
the selectin density on the substrate. At L<Lc no cell binding is detected on the
patch. At LRLc, the number of tethered cells increased with the patch length. A
theoretical model was developed to analyze our data, and accounted for the se-
lectin-mediated reversible binding kinetics, the shear-controlled association
rate and two-pathway dissociation rate. Our model successfully fits to the ex-
perimental results and, in particular, the transition of the ‘‘shear threshold phe-
nomenon’’ was correctly captured. Our model also shows that the ‘‘shear
threshold phenomenon’’ is not observed if the selectin site density is
>3000sites/mm2. In summary, our analysis has identified the minimum cell
rolling distance required for selectin-dependent binding to occur in a shear
flow. Moreover, we have developed the first analytical equation to model
cell binding with the shear threshold phenomenon.
1276-Pos
Distinguishing Binding from Allosteric Action in Escherichia Coli Phos-
phofructokinase
Bobby W. Laird, Gregory D. Reinhart.
Texas A&M Univ, College Station, TX, USA.
Thermodynamic linkage analysis involves the determination of binding param-
eters for substrate and allosteric ligand individually to free enzyme plus a cou-
pling parameter that quantifies the nature and magnitude of allosteric interac-
tion. Thus allosteric action can be differentiated from allosteric ligand and
substrate binding affinities. Previously thermodynamic linkage analysis of
the allosteric inhibition of E. coli PFK (EcPFK) by either phosphoenolpyruvate
(PEP) or its analogs implied that different functional groups within the alloste-
ric molecules play different roles in each of these two attributes. The data sug-
gested in particular that the oxygen that bridges the phosphate group to the rest
of the molecule in PEP plays a larger role in ligand binding than in allosteric
signal propagation. In an effort to continue to elucidate the role of the PEP
bridging oxygen the crystal structure of wild type EcPFK bound to PEP was
determined for the first time. S58 was found to be in position for possible in-
teractions with this bridging oxygen of PEP. EcPFK with the mutation S58A
was constructed and characterized. The modified enzyme exhibits no variation
in Km or Vmax compared to the wild type enzyme. However, the Kd for PEP is
more than 85 times that of wild type while the allosteric coupling is diminished
by 27%. The analysis of the S58A mutant provides additional evidence that the
bridging oxygen in PEP primarily contributes to the binding free energy of PEP
and makes a relatively minor contribution to the allosteric effect per se. Sup-
ported by NIH Grant GM33216 and Welch Foundation Grant A1548.
1277-Pos
Superheating of Ice in the Presence of Ice Binding Proteins
Yeliz Celik1, Laurie A. Graham2, Yee-Foong Mok2, Maya Bar3,
Peter L. Davies2, Ido Braslavsky1.
1Ohio University, Athens, OH, USA, 2Queen’s University, Kingston, ON,
Canada, 3Weizmann Institude of Science, Rehovot, Israel.
Antifreeze proteins (AFPs) are a class of ice binding proteins that are found in
many cold-adapted organisms, where they serve as inhibitors of ice crystal
growth and recrystalization. Adsorption-inhibition theory suggests that AFPs
adsorb to ice surface and surface-adsorbed AFPs should prevent ice from melt-
ing as well as from freezing and evidence for such activity was shown exper-
imentally (Knight and Devries, Science 1989). However, so far there has
been no measurement of the melting inhibition of ice in AFP solutions. We ex-
amined a series of hyperactive and moderate AFPs and measured the melting
hysteresis activity, which is defined as the elevation of the actual melting tem-
perature above the equilibriummelting point. We observed that superheated icecrystals can be held stable for hours in AFP solutions. The measured superheat-
ing values were much more appreciable for hyperactive AFPs in comparison to
moderate ones. The amount of this elevation was only a fraction of depression
the freezing. Still, in these temperatures, we measured remarkably fast melting
velocities of the superheated ice crystals which were proportional to the super-
heating values. Furthermore, we visualized fluorescently labeled AFPs on su-
perheated ice crystals. The observation of superheating of by AFPs strongly
suggest that AFPs adhere to ice surface as a part of their mechanism of action
and the binding to ice surface is irreversible.
Membrane Protein Function II
1278-Pos
Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensi-
tizes Host Cells to Apoptosis
Anke Harsman1, Michael Meinecke2, Thomas Rudel3, Joachim Rassow4,
Richard Wagner1.
1University of Osnabrueck, Osnabrueck, Germany, 2MRC Laboratory of
Molecular Biology, Cambridge, United Kingdom, 3University of Wuerzburg,
Wuerzburg, Germany, 4Ruhr-Universita¨t Bochum, Bochum, Germany.
The bacterial outer membrane porin PorB of Neisseria gonorrhoeae is an essen-
tial virulence factor of these pathogenic gram-negative bacteria which finally
lead to the apoptotic degradation of host cells. In course of the infection process
PorB is known to be transferred to the mitochondrial membranes of infected
cells where it induces the dissipation of mitochondrial membrane potential
by a so far unknown mechanism. Using single channel electrophysiology and
spectroscopic techniques we were able to reveal the molecular mechanisms un-
derlying this regulated process.
Based on a detailed study of the single channel characteristics of wildtype PorB
and the mutant PorBK98Q lacking the putative ATP-binding site, we could
identify the prerequisites for the formation of open pores in the mitochondrial
inner membrane at physiological membrane potentials. Spontaneous incorpora-
tion of the b-barrel protein into membranes was followed by using CD-spec-
troscopy revealing large structural rearrangements during membrane insertion.
We are now able to explain the molecular mechanisms by which targeting of
bacterial toxin PorB to mitochondria of infected cells is setting up host cells
to apoptosis.
1279-Pos
Functional Reconstitution of Membrane Proteins Monitored by Isother-
mal Titration Calorimetry
Nadin Jahnke1, Oxana O. Krylova1, Sandro Keller2.
1Leibniz Institute of Molecular Pharmacology (FMP), Berlin, Germany,
2Technical University Kaiserslautern, Kaiserslautern, Germany.
Membrane protein reconstitution denotes the transfer of a purified (but usually
inactive) membrane protein from detergent micelles into lipid bilayers. The aim
is to restore the native protein fold and function in a well-defined membrane
environment. The reconstitution yield critically depends on a wide range of pa-
rameters, including temperature, pH, ionic strength, as well as the type and con-
centration of detergent, lipid, protein, and additives. Moreover, it is of para-
mount importance to initiate the reconstitution process from a suitable lipid-
to-detergent ratio. Unfortunately, however, assessing the success of a reconsti-
tution experiment has thus far been limited to a trial-and-error approach, which
has substantially slowed progress in the field.
To address this problem, we have established high-sensitivity isothermal titra-
tion calorimetry (ITC) as a powerful tool for monitoring the reconstitution of
membrane proteins into lipid vesicles. Using ITC, the complex changes in
the physical state of a protein/lipid/detergent mixture during reconstitution
can be followed in a non-invasive and fully automated manner. Here we exem-
plify this approach for the prokaryotic potassium channel KcsA, which we first
purified in detergent micelles and then reconstituted into stable proteolipo-
somes at very high protein densities. Electrophysiological experiments per-
formed in planar lipid membranes confirmed that KcsA regained its functional
activity upon reconstitution.
1280-Pos
QuantitativeMeasurements of Receptor Interactions in Mammalian Cells:
Implications for Human Pathologies
Sarvenaz Sarabipour, Kalina Hristova.
The Johns Hopkins University, Baltimore, MD, USA.
Receptor Tyrosine kinases (RTKs) are family of single-pass cell membrane re-
ceptors with extracellular ligand-binding domains and intracellular kinase do-
mains, which conduct biochemical signals via lateral dimerization in the
plasma membrane. Mutations in the transmembrane (TM) domains of these re-
ceptors are known to promote unregulated signaling. An example is the
Ala391Glu mutation in the TM domain of FGFR3, which leads to pathologies


Distinguishing Binding from Allosteric Action in Escherichia Coli Phosphofructokinase 

file:///data/core-remote/dit/data/elsevier/pdf/270/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUwOTAzMTM1eA==.pdf

Distinguishing Binding from Allosteric Action in Escherichia Coli Phosphofructokinase

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector