Berezovsky, Igor N.

Mitternacht, Simon

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Monday, February 27, 2012 225aof dynamics in the specificity of cytochrome P450 using 2D IR spectroscopy. In
efforts toward extending the 2D IR studies beyond heme proteins, an azidopheny-
lalanine vibrational probe was introduced at a specific site withinmyoglobin using
orthogonal transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs evolved to in-
sert azidophenylalanine in response to an amber codon. The azidophenylalanine
vibrational probe was placed within the heme pocket at Phe43, permitting investi-
gationof one site inmyoglobinvia twodifferent vibrational probes - the genetically
introduced azidophenylalanine and heme bound CO. The use of two probes
allowed assessment of the perturbation due to introduction of each and the mea-
surement of the heme pocket dynamics via two perspectives, showing that the re-
ported dynamics were reflective of the protein motions, and not intrinsic to
a particular probe. This approach can be used in the future to characterize the
dynamics at virtually any site throughout virtually any protein.
1135-Plat
A DNA Lesion Alters the Global Conformational Dynamics of a Y-Family
DNA Polymerase during Catalysis
Brian A. Maxwell.
The Ohio State University, Columbus, OH, USA.
8-Oxo-7,8-dihydro-20-deoxyguanine (8-oxoG), a major oxidative DNA lesion, can
lead to genomic mutations if it is bypassed unfaithfully by DNA polymerases in
vivo. However, our pre-steady state kinetic studies showed that DNA polymerase
IV (Dpo4), a prototype Y-family enzyme from Solfolobus solfataricus, bypassed
8-oxoG both efficiently and faithfully. For the first time, our stopped-flow Fo¨rester
resonance energy transfer (FRET) studies revealed that a DNA polymerase altered
its synchronized, globalconformationaldynamics in response to aDNAlesion.Rel-
ative tonucleotide incorporation intoundamagedDNA, threeof the fourdomainsof
Dpo4 undertook different conformational transitions during 8-oxoGbypass and the
subsequent extension step. Moreover, the rapid translocation of Dpo4 along DNA
induced by nucleotide binding was significantly hindered by the interactions be-
tween embedded8-oxoGandDpo4during the extension step. These results unprec-
edentedly demonstrate that a Y-family DNA polymerase employs different global
conformational dynamics when replicating undamaged and damaged DNA.
1136-Plat
Bayesian Modeling of Crystallographic Disorder
Timothy Fenn1, Michael Schnieders2, Vijay Pande1.
1Stanford University, Stanford, CA, USA, 2University of Texas, Austin,
Austin, TX, USA.
Macromolecular crystallography is typified by a ‘‘static’’ crystal structure that re-
sults from the experimental refinement. However, we posit that the single atomic
model is amyopic representation of the statistical ensemble, and derive theBayes-
ian logic to illustrate a rigorous, probabilistic
representation of the data using a model en-
semble. Using ubiquitin and a complex of
a viral entry protein bound to its cellular re-
ceptor as examples, we will show the utility
of this method in describing biological func-
tion and protein dynamics in the context of
X-ray crystallography that was previously
not possible. Finally, a brief description of
the software with which this is being accom-
plished will be presented.1137-Plat
Role of Electrostatics in the Mechanochemical Rotary Mechanism of
F1-ATPase
Shayantani Mukherjee, Arieh Warshel.
University of Southern California, Los Angeles, CA, USA.
F1-ATPase is a remarkablemotor consisting of a rotating stalk and a stator crown
undergoing catalysis. The enzyme synthesizes or hydrolyzes ATP depending on
the clockwise or anticlockwise rotation of the stalk respectively. InnovativeAFM
and single molecule spectroscopy have led to the direct observation of interme-
diate events during the hydrolysis cycle (1), although a consistent theoretical un-
derstanding is still missing that successfully bridges the gap between modeling
andobserved facts.Hereweuse a coarse grained (CG)modelwith a realistic elec-
trostatic treatment (2) to dissect the rotary mechanism of the complete F1-
ATPase motor. The structure-based free energy landscape of the enzyme gener-
ated by ourCGmodel reproduced the energetics of themechanochemical process
(3), thus providing a theoretical understanding of the tight coupling between the
key chemical and conformational events. It is found that the landscape along the
observed rotary path are consistentwith the singlemolecule experiments and also
support the baffling experimental finding that the stalk rotates in the right direc-
tion even in the absence ofmost of the part embeddedwithin the crown cavity (4).
These results not only highlight the importance of electrostatics in guiding the
action of F1-ATPase, but also provide valuable means to discriminate betweendifferent theoreticalmodels like stericmechanism, elastic strain basedmechanism
or stochastic Brownian motor underlying the action of biological rotary motors.
(1) Adachi K, et al. Cell, 130, 309–321 (2007).
(2) B. M. Messer, M. Roca,Z. T. Chu, S.Vicatos, A. V. Kilshtain and A. Warshel,
PROTEINS,78, 1212–1227 (2010).
(3) S. Mukherjee and A. Warshel , PNAS submitted (2011).
(4) Furuike S, et al. Science, 319, 955–958 (2008).
1138-Plat
Detection of Allosteric Signal Transmission by Information-Theoretic
Analysis of Protein Dynamics
Alessandro Pandini1,2, Arianna Fornili1, Franca Fraternali1, Jens Kleinjung2.
1King’s College London, London, United Kingdom, 2MRC - National
Institute for Medical Research, London, United Kingdom.
Introduction
Allosteric inhibitors were shown to be effective in targeting multiple conforma-
tional states and more selective than competitive inhibitors [1]. However, allo-
steric signal transmission is difficult to model because often mediated by small
local changes propagating along multiple pathways in the protein structure [2].
Therefore, we developed a method to identify these pathways by an
information-theoretic analysis of Molecular Dynamics (MD) simulations.
Methods
First, MD ensembles are mapped onto a canonical set of representative frag-
ments, i.e. a set of discrete 4-residue states [3]. By this mapping, the contribu-
tion of rigid fragment roto-translations is removed and relevant changes are
detected, even if subtle. Second, local motions are modelled as transitions be-
tween the fragment states. This removes background noise from harmonic
high-frequency fluctuations. Third, the communication between parts of the
molecule is modelled by the coupling of fragment transitions. These are re-
solved spatially and temporally. The time-averaged spatial couplings are ana-
lyzed with a network model of fragment correlations. Then communication
pathways are characterised by time-resolved analysis of fragment couplings.
Results
The method was applied to the NtrC, CheY, and FixJ receiver domains. The
results are consistent with the emerging picture of allosteric regulation [2]:
a pre-organized network of fragment couplings connects the allosteric and
functional sites already in the inactive state, and fragments with high network
centrality (hubs) are part of functionally relevant regions. Critical nodes on the
signalling pathways correspond to residues experimentally known to affect the
kinetics and thermodynamics of the allosteric mechanism.
References
1. Kar, G., Keskin, O., Gursoy, A. & Nussinov, R. Curr Opin Pharmacol 10,
715–22 (2010).
2. del Sol, A., Tsai, C.J., Ma, B.&Nussinov, R. Structure 17, 1042–1050 (2009).
3. Pandini, A., Fornili, A., Kleinjung, J. BMC Bioinformatics 11, 97 (2010).
1139-Plat
Binding Leverage and Leverage Coupling as Determinants of Allosteric
Regulation and Communication
Igor N. Berezovsky, Simon Mitternacht.
University of Bergen, Bergen, Norway.
Allosteric regulation involves collective conformational transitions or fluctua-
tions between a few closely related states, caused by the binding of effector mol-
ecules [1]. The states involved are inherent to the protein, in the sense that they are
visited by the protein also in the absence of effector ligands. We developed the
measure binding leverage [2] to find sites where ligand binding can shift the con-
formational equilibrium of a protein. Binding leverage is calculated for a set of
motion vectors representing independent conformational degrees of freedom.
We analyze single catalytic domains and multimeric allosteric enzymes with
complex regulation. For the majority of the analyzed proteins, we find that both
catalytic and allosteric sites have high binding leverage. To analyze allosteric
communication between binding sites, we introduce the concept of leverage cou-
pling [3], based on the assumption that only pairs of sites that couple to the same
conformational degrees of freedom can be allosterically connected. We demon-
strate how leverage coupling can be used to analyze allosteric communication
in a range of enzymes (regulated by both ligand binding and post-translational
modifications) and huge molecular machines such as chaperones. Because bind-
ing leverage and leverage coupling can be calculated from a single crystal struc-
ture it should be a useful tool for characterizing proteins of unknown function and
predicting latent allosteric sites in any protein, with implications for drug design.
1.Mitternacht, S.&Berezovsky, I.N. 2011.Ageometry-basedgeneric predictor for
catalytic and allosteric sites. Protein Engineering, Design&Selection 24, 405–409.
2. Mitternacht, S. & Berezovsky, I.N. 2011. Binding leverage as a molecular
basis for allosteric regulation. PLoS Comp Biol 7, e1002148.
3. Mitternacht, S. & Berezovsky, I.N. 2011. Coherent conformational degrees
of freedom as a structural basis for allosteric communication, Submitted.


Binding Leverage and Leverage Coupling as Determinants of Allosteric Regulation and Communication 

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Binding Leverage and Leverage Coupling as Determinants of Allosteric Regulation and Communication

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