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WSN: Biophysical Journal (vol.69, number 1, Jul-95)
(from URL: http://biosci.cbs.umn.edu/biophys/bj/)
Electrostatic and Hydrodynamic Orientational Steering Effects in
Enzyme-Substrate Association
Jan Antosiewicz ** and J. Andrew McCammon
Departments of Chemistry and Biochemistry, and Pharmacology, University of
California at San Diego, La Jolla, CA 92093-0365
**On leave from Department of Biophysics, University of Warsaw, Poland
ABSTRACT
Diffusional encounters between a dumbbell model of a cleft enzyme and a
dumbbell model of an elongated ligand are simulated by Brownian dynamics.
The simulations take into account electrostatic and hydrodynamic
interactions between the molecules. It is shown that the primary effect of
inclusion of hydrodynamic interactions into the simulation is an overall
decrease in the rate constant. Hydrodynamic orientational effects are of
modest size for the systems considered here. They are manifested when
changes in the rate constants for diffusional encounters favoured by
hydrodynamic interactions are compared with those favoured by electrostatic
interactions as functions of the overall strength of electrostatic
interactions. The electrostatic interactions modify the hydrodynamic torques
by modifying the drift velocity of the substrate toward the enzyme. We
conclude that simulations referring only to electrostatic interactions
between an enzyme and its ligand may yield rate constants that are somewhat
(e.g. 20 %) too high, but provide realistic descriptions of the
orientational steering effects in the enzyme-ligand encounters.
Protonation Dynamics of the alpha-Toxin Ion Channel from Spectral
Analysis of pH Dependent Current Fluctuations
John J. Kasianowicz1 and Sergey M. Bezrukov2,3
1) National Institute of Standards and Technology, Biotechnology
Division, Biosensors Group, 222/A353, Gaithersburg, MD 20899
2) National Institutes of Health, Laboratory of Structural Biology, DCRT,
Building 5, Room 405, Bethesda, MD 20892-0580
3) St. Petersburg Nuclear Physics Institute of the Russian Academy of Sciences,
Gatchina, Russia 188350
Abstract
To probe protonation dynamics inside the fully open a-toxin ion
channel, we measured the pH-dependent fluctuations in its current. In the
presence of 1M NaCl dissolved in H2O and positive applied potentials
(from the side of protein addition), the low frequency noise exhibited a
single well-defined peak between pH 4.5 and 7.5. A simple model in which
the current is assumed to change by equal amounts upon the reversible
protonation of each of N identical ionizable residues inside the channel
describes the data well. These results, and the frequency dependence of
the spectral density at higher frequencies, allow us to evaluate the
effective pK = 5.5, as well as the rate constants for the reversible
protonation reactions: kon = 8x109 M-1 s-1 and koff = 2.5x104 s-1. The
estimate of kon is only slightly less than the diffusion-limited values
measured by others for protonation reactions for free carboxyl or
imidazole residues.
Substitution of H2O by D2O caused a 3.8-fold decrease in the dissociation
rate constant and shifted the pK to 6.0. The decrease in the ionization rate
constants caused by H2O/D2O substitution permitted the reliable measurement
of the characteristic relaxation times.
Filipin Fluorescence Quenching By Spin-Labelled Probes.
Studies In Aqueous Solution And In A Membrane Model System.
Miguel Castanho 1,2 And Manuel Prieto 1,*
1 Centro de Quimicia-Fisica Molecular, Instituto Superior Tecnico, 1096
Lisboa Codex, Portugal. 2 Departamento de Quimica e Bioquimica, Faculdade
de Ciencias da universidade de Lisboa, R. Ernesto Vasconcelos, Bloco C1-5
1700 Lisboa, Portugal.
ABSTRACT
A detailed photophysical study of the fluorescence quenching (transient
and steady state) of the macrolide antibiotic filipin by nitroxide
substituted fatty acids and a cholesterol derivative was carried out,
aimed at determining its transverse position in a model system of
membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine,
DPPC1). Filipin partitionates efficiently into membranes (Kp =3D
(5.0=B11.0).103; 20=BAC) and it was concluded that the antibiotic is buried
in the membrane, away from the lipid-water interface. In addition,
information on the organization of the quenchers was also obtained. The
5-nitroxide derivative of the fatty acid in essentially randomly
distributed, while the 16-nitroxide is aggregated at concentrations
higher than ca. 5% molar. For the cholesterol compound the results point
out to a phase separation at concentrations higher than 3% molar (below
this limit concentration filipin associates with the derivatized sterol
with KA =3D 20 M-1, assuming a 1:1 interaction). We propose that this phase
separation and the aggregation state of filipin in the aqueous solution,
may be key processes in the antibiotic mode of action. A systematic and
general approach to fluorescence quenching data analysis in complex (e.
g. biochemical) systems is also presented.
Solvent Effect on Phosphatidylcholine Headgroup Dynamics as Revealed by the
Energetics and Dynamics of Two Gel-State Bilayer Headgroup Structures at
Subzero Temperatures
Chang-Huain Hsieh and Wen-guey Wu
Institute of Life Sciences, National Tsing Hua University Hsinchu, Taiwan
30043
ABSTRACT The packing and dynamics of lipid bilayers at the phosphocholine
headgroup region within the temperature range of -40oC to -110oC have been
investigated by solid state NMR measurements of selectively
deuterium-labeled H2O/dimyristoylphosphatidylcholine (DMPC) bilayers. Two
coexisting signals with 2H NMR quadrupolar splittings of 36.1 and 9.3 (or
smaller) KHz were detected from the -CD3 of choline methyl group. These two
signals have been assigned to two coexisting gel-state headgroup structures
with fast rotational motion of -CD3 and -N(CD3)3 group, respectively, with a
three fold symmetry. The largest quadrupolar splitting of the NMR signal
detected from the -CD2 of Calpha and Cbeta methylene segment was found to be
115.2 KHz, which is 10% lower than its static value of 128.2 KHz. Thus,
there are extensive motions of the entire choline group of gel-state
phosphatidylcholine bilayers even at a subzero temperature of -110oC. These
results strongly support the previous suggestion (Dufourc et al., 1992,
Biophys. J. 61, 42-57) that 31P chemical shift tensor elements of DMPC
determined under similar conditions are not the rigid static values. The
free energy difference between the two gel-state headgroup structures were
determined to be 26.3 +/- 0.9 KJ/mol for fully hydrated bilayers.
Furthermore, two structures with similar free energy difference were also
detected for "frozen" phosphorylcholine chloride solution in a control
experiment, leading to the conclusion that the two structures may be
governed solely by the energetics of fully hydrated phosphocholine
headgroup. The intermolecular interactions among lipids, however, stabilize
the static headgroup structure as evidenced by the apparently lower free
energy difference between the two structures for partially hydrated lipids
bilayers. Evidence is also presented to suggest that one of the headgroup
structures with trimethylammonium group rotation, which is not compatible to
the static headgroup structure in crystals, is due to the dielectric
relaxation of the slowly reorienting interbilayer water molecules near the
physical edge of membrane surface. Finally a molecular model of the
hydration-induced conformational changes at the torsion angle alpha5 of
O-C-C-N+ bond is proposed to explain the two detected coexisting headgroup
structures. These results emphasize the important role of trimethylammonium
group in monitoring the structure and dynamics of lipid headgroup.
A New View of Water Dynamics in Immobilized Proteins
Bertil Halle and Vladimir P. Denisov
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