Claycomb, James R.

Infante, Hans L.

Miller, John H.

Vajrala, Vijayanand

Elsevier - Publisher Connector 

Torque Generation Mechanism of ATP Synthase and Other Rotary Motors 

8a Sunday, March 1, 2009quantified by surface plasmon resonance. NDPK-D was also able to cross-link
anionic phospholipid-containing liposomes as seen in light scattering assays,
suggesting that the hexameric kinase could promote intermembrane contacts.
Mutation of the central arginine (R90) in a surface exposed basic RRK motif
unique to NDPK-D strongly reduced these membrane interactions. In a model
using HeLa cells naturally almost devoid of NDPK-D, wt protein and R90D
mutant were stably expressed, but only wt protein was found attached to mem-
branes. Respiration was significantly stimulated by the NDPK substrate TDP
only in mitochondria containing wt NDPK-D, but not in those expressing
R90D mutant that is catalytically equally active. This indicates local ADP re-
generation in the mitochondrial intermembrane space and a tight functional
coupling of NDPK-D with oxidative phosphorylation that depends on the mem-
brane-bound state of the kinase. A model is proposed for a mitochondrial
NDPK microcompartment.
39-Plat
Using Two-photon Excited Fluorescence Intensity- and Lifetime-based
NADH Imaging to Investigate Cochlea Metabolism
LeAnn M. Tiede, Jorge A. Vergen, Clifford Hecht, Richard Hallworth,
Michael G. Nichols.
Creighton University, Omaha, NE, USA.
Metabolism and mitochondrial dysfunction are thought to be involved in many
different hearing disorders including noise induced hearing loss and presbycu-
sis. We have employed two-photon fluorescence imaging of intrinsic mitochon-
drial reduced nicotinamide adenine dinucleotide (NADH) to study the meta-
bolic status of the different cell types in excised yet intact mouse organ of
Corti preparations. Recent published studies employ fluorescence lifetime im-
aging (FLIM) to determine the ratio of the free to enzyme-bound fluorophores
populations that occur during changes in metabolism. We have compared tra-
ditional intensity based methods to FLIM in order to evaluate the two different
methods in both cultured cells and the excised organ of Corti. Treatment with
both metabolic uncouplers and inhibitors caused systematic shifts in both the
lifetime and populations of the free and bound pools of NADH, resulting in sig-
nificant differences in the calculated concentration of NADH when compared
to using intensity alone to calculate the same value. Mapping of the locations
of the individual lifetimes, shows that the lifetime of NADH varies in different
cellular locations as well as in different cell types. Possible implications for the
study of hearing loss will be discussed.
Supported by NIH DC 08895, NIH P20 RR016475 from the INBRE
Program of the National Center for Research Resources., and NIH R15-
GM085776.40-Plat
Mitochondrial Energy Metabolism and Ca2þ Handling in Pancreatic
Beta-cells. A System Analysis Approach
Leonid E. Fridlyand, Louis H. Philipson.
University of Chicago, Chicago, IL, USA.
Pancreatic islet beta-cells respond to rising blood glucose by increasing oxida-
tive metabolism, leading to both an increased mitochondrial membrane poten-
tial (Jm) and ATP/ADP ratio in cytoplasm. This leads to a closure of KATP
channels, depolarization of the plasma membrane, influx of calcium and the
eventual secretion of insulin. Such a signaling mechanism suggests that mito-
chondrial metabolism and ATP/ADP ratio regulation in beta-cells may be spe-
cially coupled in comparison with other cell types. We performedmathematical
modeling to quantitatively assess how cytoplasmic ATP/ADP ratio can be
controlled by mitochondria. The cytoplasmic part of the model includes gluco-
kinase, glycolysis, pyruvate reduction, NADH and ATP production and
consumption. The mitochondrial part of the model includes production of
NADH, which is regulated by pyruvate dehydrogenase. NADH is used in the
electron transport chain to establish a proton motive force, driving the F1F0-
ATPase. Mitochondrial matrix Ca2þ is determined by the Ca2þ uniporter and
Naþ/Ca2þ exchanger. The model is described by ordinary differential equations
for cytoplasmic and mitochondrial parameters. The model simulates the re-
sponse of the ATP/ADP ratio to changes in substrate delivery, to inhibition
of the mitochondrial Ca2þ exchanger and other effects. We found that mito-
chondrial Jm should be in a range lower than 150 mV (where F1F0-ATPase
is sensitive to Jm) to provide a sensitivity of the ATP/ADP ratio to glucose
in beta-cells. On other hand,Jm can work in the range above 150 mV to pro-
vide a maximal F1F0-ATPase productivity in other cell types (for example in
myocytes). Kinetic analysis of the model reveals that these differences can
be simulated by a decreased respiratory activity and higher leak capacity in
beta-cell mitochondria in comparison with muscle cell mitochondria that
were found recently by Affourtit and Brand (2006).41-Plat
Modeling Regulation of Mitochondrial Free Ca2þ by ATP/ADP-Depen-
dent Ca2þ Buffering
Ranjan K. Dash, Matthew D. Thompson, Kalyan C. Vinnakota,
Johan Haumann, Mohammed Aldakkak, Amadou K.S. Camara,
David F. Stowe, Beard A. Daniel.
Medical College of Wisconson, Milwaukee, WI, USA.
Introduction: Mitochondrial free [Ca2þ] ([Ca2þ]m) is regulated by cation
fluxes through the Ca2þ uniporter (CU), Naþ/Ca2þ exchanger (NCE), Naþ/
Hþ exchanger (NHE), and Ca2þ/Hþ exchanger (CHE) as well as via Ca2þ buff-
ering by the mitochondrial proteins. However, the regulation of [Ca2þ]m via
ATP/ADP-dependent dynamic Ca2þ buffering mechanism inside the mito-
chondrial matrix during transient state-3 respiration is not well known.
Methods: To gain a quantitative understanding of this Ca2þ buffering phenom-
enon, we developed a computational model of mitochondrial bioenergetics and
Ca2þ handling by integrating our recent biophysical models of the CU, NCE,
NHE, and CHE into our well-validated model of mitochondrial oxidative phos-
phorylation, TCA cycle, and electrophysiology. The model also accounts for
binding and buffering of cations with metabolites, including ATP, ADP and
Pi. Experiments were performed to spectrofluorometrically measure [Ca2þ]m,
pHm, membrane potential (DJm), and NADH redox state in guinea pig heart
mitochondria suspended in Naþ and Ca2þ free buffer medium (ensured with
~50 mM of EGTA) with 0.5 mM pyruvic acid (HPyr). Dynamics were inferred
with various addition of CaCl2 (0, 10, 25 mM of CaCl2; 16, 88, 130 nM of free
[Ca2þ] followed by 250 mMof ADP in the presence or absence of carboxyatrac-
tyloside (ANT blocker) and oligomycin (F1F0-ATPase blocker). Results and
Discussion: Model analysis of the data on (i) initial decrease of [Ca2þ]m
with addition of Naþ-independent substrate HPyr, and (ii) transient increases
of [Ca2þ]m with addition of ADP suggests ATP/ADP-dependent dynamic
Ca2þ buffering inside the cardiac mitochondrial matrix. This model will be
helpful to understand mechanisms by which [Ca2þ]m both regulates, and is
modulated by, mitochondrial energy metabolism.
42-Plat
The External Stalk of the FoF1-ATPase: 3D-Structure of the b-Dimer
Oleg A. Volkov, Susan J. Pandey, John G. Wise, Pia D. Vogel.
Southern Methodist University, Dallas, TX, USA.
The structure of the external stalk of the FoF1-ATP synthase and its function
during catalysis remain one of the important questions in bioenergetics.
Proteomics, structure prediction, molecular modeling and ESR spectroscopy
using site-directed spin labeling were employed to elucidate the structure and
interfacial packing of the E. coli b-subunit homodimeric and Synechocystis
bb’ heterodimeric stalks of ATP synthases.
b-Subunits of different origin demonstrate little sequence similarity. Structure
prediction algorithms, however, showed that all of the compared sequences
contain extensive heptad repeats, suggesting that these proteins may favorably
pack as left-handed coiled coils (LHCC).
Molecular modeling of homo- and heterodimeric b produced low energy
LHCC. Extensive mutagenesis followed by site-directed spin labeling and sub-
sequent ESR investigations in soluble homo- and heterodimeric b-constructs
allowed the determination of inter- and intra-subunit distances.
Inter-spin distances obtained by ESR agreed very well with distances derived
from LHCC molecular models of b- and bb’-dimers and therefore strongly sup-
port our proposition that dimeric external stalks of ATP synthases indeed form
left-handed coiled coils.
The extreme C-terminal part of the b-dimer is not predicted to form a coiled
coil structure. We are presently investigating this part of the second stalk
both when in solution and when in complex with soluble F1-ATPase. The in-
fluence of subunit d is of particular interest due to its proposed direct interaction
with the C-terminus of subunit b.
Initial site-directed spin labeling and ESR experiments using complete E. coli
FoF1-ATP synthase indicate that the inter-subunit packing of the b-dimer
changes during catalytic turnover, which may be a mechanism for elastic cou-
pling of the different rotating parts of the enzyme.
43-Plat
Torque Generation Mechanism of ATP Synthase and Other Rotary
Motors
John H. Miller1, Vijayanand Vajrala1, Hans L. Infante1,
James R. Claycomb2.
1University of Houston, Houston, TX, USA, 2Houston Baptist University,
Houston, TX, USA.
Ion driven rotary motors, including F0-ATP synthase (F0) and the bacterial fla-
gellar motor, convert energy from ion translocation into torque and rotary
Sunday, March 1, 2009 9amotion. Here we propose a mechanism whereby electric fields emanating from
channels in the stator(s) create forces acting on ion binding sites in the rotor,
driving it to rotate. The model predicts a scaling law relating torque to ion mo-
tive force and number of stators, consistent with experiment. The rotor of F0
drives the gamma-subunit to rotate within the ATP-producing complex (F1),
working against an opposing torque that rises and falls periodically with angu-
lar position. Drawing an analogy with the washboard potential of the supercon-
ducting Josephson junction, we compute ATP production rate vs. ion motive
force, finding excellent quantitative agreement with experiment and accounting
for the crossover from ATP production to consumption. Plausible mechanisms
for reversing the direction of bacterial flagellar rotation become self-evident in
the model. (Supported by NIH R21CA133153, TcSUH, Welch (E-1221), and
THECB-ARP.)Platform D: RNA Folding & Ribosome
44-Plat
Laser Assisted Single-molecule Refolding
Rui Zhao, Elvin Alema´n, David Rueda.
Wayne State University, Detroit, MI, USA.
In vivo, many RNA molecules can adopt multiple conformations depending on
their biological context. For example, an RNA molecule initially in a stable
hairpin conformation may later interact with a second RNAmolecule, thus trig-
gering a dimerization reaction. This is the case of the HIV Dimerization Initi-
ation Sequence (DIS) and the DsrA RNA in bacteria. It is quite common that
the initial interaction between the two RNAs takes place via complementary
unpaired regions, forming a so-called kissing complex. However, the exact
kinetic mechanism by which the two RNA molecules reach the dimerized state
is still not well understood.
To investigate the refolding energy surface of RNA molecules, we have devel-
oped new technology based on the combination of single molecule spectros-
copy with laser induced temperature jump kinetics, called Laser Assisted
Single-molecule Refolding (LASR). LASR enables us to induce folding reac-
tions of otherwise kinetically trapped RNAs at the single molecule level, and
to characterize their folding landscape. Single molecule time trajectories
show that we can drive the dimerization reaction between two trapped kissing
RNA hairpins with LASR and use this data to calculate folding enthalpies and
entropies. Our LASR experiments indicate that the RNA kissing complex is
a stable intermediate state that facilitates the dimerization reaction.
LASR provides an exciting new approach to study molecular memory effects
and kinetically trapped RNAs in general. LASR is readily applicable to study
DNA and protein folding as well.
45-Plat
Single Molecule Analysis of Group I Ribozyme Folding Reveals Pro-
nounced Ruggedness Throughout Its Folding Landscape
Sergey Solomatin1, Max Greenfeld1, Steven Chu2, Dan Herschlag1.
1Stanford University, Stanford, CA, USA, 2University of California,
Berkeley, Berkeley, CA, USA.
It is well established that biological activity of macromolecules is intimately
related to their structures. Significant efforts are directed at characterizing
macromolecular structures and structure-function relationships. It is less
appreciated that because of their complexity macromolecules are likely to
fold not into unique structures, but into ensembles of different conforma-
tions distinct in their biological activity. Strong evidence for such complex
behavior has been recently obtained in several macromolecular systems,
mostly by single molecule methods. Understanding biological activity of
macromolecules requires detailed characterization of the species that co-ex-
ist in the ‘‘folded’’ states, in other words - mapping of their folding energy
landscape.
We used single molecule FRET to map folding energy landscape of a catalytic
RNA - ribozyme derived from a group I intron. Labeling several different po-
sitions within the ribozyme allowed us to probe different parts of the folding
landscape: from essentially unfolded to fully folded ribozyme. These experi-
ments revealed broad heterogeneity of folding behaviors of individual mole-
cules. Strikingly, even in conditions in which the ribozyme is fully folded
and active, different conformations retain distinct activity. Distinct folding be-
haviors are ‘‘remembered’’ by individual molecules upon long incubation in
conditions in which the ribozyme lacks most of its tertiary structure. This find-
ing suggests that the origins of heterogeneity might appear at the level of sec-
ondary structure. Pinning down the structural basis for ruggedness of RNA
folding landscapes and understanding its role in biology represents an exiting
future challenge.46-Plat
Identifying Energy Barriers in RNA Folding Through Kinetic Model
Enumeration
Joshua S. Martin1, Joerg Schlatterer2, Michael Brenowitz2,
Alain Laederach1.
1Computational and Structural Biology, Wadsworth Institute, Albany, NY,
USA, 2Department of Biochemistry, Albert Einstein College of Medicine,
Bronx, NY, USA.
Large RNA molecules are known to fold through multiple parallel pathways to
achieve their functional conformation. These pathways include stable interme-
diate structures that we identify using our KinFold algorithm (http://simtk.org/
home/kinfold) for the analysis of time-resolved hydroxyl radical footprinting
experiments. KinFold enumerates kinetic model topologies to determine the
structure, lifetimes, and abundance of intermediates along the folding pathways
of RNA. We are therefore able to identify the best fitting kinetic model for each
folding reaction studied. We used this approach to study the folding of the L-21
T. thermophila group I intron, a catalytic ribozyme, at 6 temperatures ranging
from 25 to 51 C. Our analysis of this data reveals that the rates for a majority
of the transitions (between intermediate, folded and unfolded states) obey the
Arrhenius equation, allowing us to estimate the relative energies of activation
for inter conversion between the states of an RNA. Furthermore, we are able to
estimate the lifetimes of the different intermediate structures. These results re-
veal a simple landscape, where folding of the peripheral elements of the RNA
create significant barriers to folding. Our approach is unique in that it allows us
to simultaneously estimate the activation energies for all transitions in an RNA
folding reaction.
47-Plat
Uncovering The Mechanism Used By Drosophila Snf Protein To Specifi-
cally Bind Two RNAs
Kathleen B. Hall, Gregory DeKoster, Sandra Williams.
Washington University School of Medicine, Saint Louis, MO, USA.
The Drosophila SNF protein is a natural chimeric protein that evolved from
the metazoan U1A and U2B’’ proteins. In the fly, SNF is found in both the
U1 and U2 snRNPs, where it is assumed to bind to the U1 snRNA Stemloop
II (the site of U1A binding) and U2 snRNA Stemloop IV (the site of U2B’’
binding), respectively. Like U1A and U2B’’ proteins, SNF has two RNA Rec-
ognition Motifs (RRM). By analogy to U1A protein, the N-terminal RRM
should bind RNA, while the C-terminal RRM does not. Our experiments
show that indeed the SNF C-terminal RRM does not bind RNA; NMR data
show it to be soluble and stable. The SNF N-terminal RRM alone is not stable,
however, and requires the C-terminal domain to maintain a folded form. NMR
experiments and 15N-relaxation data show that the N-terminal RRM un-
dergoes conformational exchange on the chemical shift timescale, while the
C-terminal RRM has uniformly high order parameters characteristic of a
more rigid protein.
We hypothesize that the increased flexibility of the N-terminal RRM is part of
its RNA binding mechanism, since SNF does indeed bind both U1 snRNA
Stemloop II and U2 snRNA Stemloop IV. The affinity of SNF for these two
RNAs differs by three orders of magnitude, indicating that one is preferentially
bound. By comparison, however, the affinities of the human U1A protein for
these two RNAs differ by more than 106-fold, indicating that SNF has found
a way to bind both RNAs. This hypothesis will be tested by mutation of the
N-terminal domain, with subsequent determination of protein structure, dynam-
ics, and RNA binding.
48-Plat
Parallel Pathways in 30S Ribosome Assembly
WilliamK. Ridgeway, Zahra Shajani, David P. Millar, James R. Williamson.
The Scripps Research Institute, La Jolla, CA, USA.
The bacterial 30S ribosomal subunit self-assembles in vitro to form an 850kDa
RNP. Assembly is generally thought to advance when the core 16S rRNA nav-
igates through many low-energy kinetic traps, guided by the 20 small-subunit
proteins that recognize and lock in native RNA tertiary structure. Kinetic
analyses of protein binding reveal a highly choreographed, ordered assembly
process, consistent with large-scale qualitative observations of ordered protein
binding and measurements of thermodynamic cooperativity within synthetic
fragments of the 30S. However, the precise order of events in this complex pro-
cess are not well understood.
We have constructed a 2-photon, 3-color detection Fluorescence Correlation
Spectroscopy (FCS) microscope to measure the kinetic cooperativity in the
assembling 30S ribosome. Up to three ribosomal proteins of interest can be la-
beled with different fluorophores, and the populations of up to seven different
bound species with different combinations of labels can be discriminated by


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Torque Generation Mechanism of ATP Synthase and Other Rotary Motors

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