Grubmuller, Helmut

Grubmüller, H.

MPG.PuRe

Dynamics of the ribosomal subunit Interface during TRNA translocation at near-atomic resolution.

Elsevier - Publisher Connector 

Dynamics of the Ribosomal Subunit Interface during TRNA Translocation at Near-Atomic Resolution 

4a Saturday, February 2, 2013to actin, whereas fast myosins such as muscle myosin-2 and some class-1myosin members have a low duty ratio (< 0.1). The members of the respectivegroups are assumed to use different ways to couple conformationalchanges at the nucleotide binding regions to changes that occur at the actinbinding sites. Conserved active-site elements termed switch-1 and switch-2play a major role in the coupling mechanism; however, whether and to whichextent small variations in the sequence of switch-1 and switch-2 affect the dutyratio of a given myosin, thus determining its ability for processive movement,fast contractility or tension bearing remains unresolved. Based on structuralconsiderations and confirmed by mutational analyses, we identified keyresidues in the nucleotide binding pocket that are responsible for makingADP dissociation kinetics dependent on the concentration of free magnesiumions. The exchange of a single amino acid in switch-2 affected the motor prop-erties of all myosins tested, but also transformed low duty ratio motors into highduty ratio and vice versa. In addition, x-ray structural analyses and molecularmodeling allowed us to relate the observed changes to altered coupling betweenthe active sites and actin binding regions. These results, together with our cellbiological studies demonstrating for the first time that magnesium ions havea regulatory role on motor protein function in vivo reveal that, switch-2 canact as magnesium sensor critically determining the mechanochemical proper-ties of myosins.22-SubgFrom Single Molecule Fluctuation to Muscle Contraction: A BrownianModel of A.F. Huxley’s HypothesesToshio Yanagida1,2, Lorenzo Marcucci1, Mitsuhiro Iwaki2.1University of Osaka, Japan, 2Riken QBiC, Osaka, Japan.Adaptive force generation of muscle in response to external stimuli is the resultof thermally fluctuating, cyclical interactions between myosin and actin, whichtogether form the actomyosin complex. Normally, these fluctuations are mod-elled using transition rate functions that are based on muscle fiber behaviour, ina phenomenological fashion (Huxley , 1957; Huxley & Simmons, 1971). How-ever, such a basis reduces the predictive power of these models. As an alterna-tive, we propose a model which uses direct single molecule observations ofactomyosin fluctuations (Kitamura, et al. Nature 1999, BIOPHYSICS, 2005;Iwaki et al. Nat. Chem Biol. 2009). We precisely estimate the actomyosinpotential bias and use diffusion theory to obtain a Brownian ratchet modelthat reproduces the complete cross-bridge cycle. The model is validated bysimulating several macroscopic experimental conditions, while its interpreta-tion is compatible with two different force-generating scenarios (Lorenzo &Yanagida, PloS One, 2012).23-SubgWatching Individual DNA Helicases and Motor Proteins Behaving andMisbehavingStephen Kowalczykowski.Microbiology, University of California, Davis, Davis, CA, USA.We can now watch individual proteins acting on single molecules of DNA.Using these approaches, we have been imaging the translocation of motor pro-teins, such as the ssDNA translocases and DNA helicases, RecBCD, RecQ, andSgs1, and the dsDNA translocases and chromatin-remodeling enzymes, Rad54and Tid1 proteins.The RecBCD enzyme comprises two motor subunits: RecB, a 3’/5’ SF1-helicase, and RecD, a 5’/3’ SF1-helicase. Although the mean unwindingrate of individual RecBCD enzyme molecules is close to ensemble measure-ments, the rates of individual enzyme molecules vary widely. IndividualRecBCD molecules unwind DNA at constant rates, suggesting static, notdynamic, disorder. This apparent conformational heterogeneity is static onthe experimental time scale of DNA unwinding. We discovered that transientlyhalting a single enzyme-DNA complex changes the rates of the RecBCD mol-ecule. We will demonstrate that the behavior of individual RBCD enzymesmanifests a basic tenet of the ergodic hypothesis, suggesting that ligand bindingkinetically traps a single conformer that is capable of redistributing to all otherstates within the population of molecules at equilibrium.Using TIRF microscopy, the helicase activity of RecQ was visualized on singlemolecules of DNA using a fluorescent ssDNA sensor. By monitoring the for-mation and progression of individual unwinding forks, we observe that boththe frequency of initiation and rate of unwinding are highly dependent onRecQ concentration. We establish that unwinding forks can initiate internallyby melting into dsDNA and can proceed in both directions. The findings sug-gest that initiation requires a RecQ dimer, and continued unwinding involvesthe repeated cooperative action of multiple monomers at the DNA unwindingfork. We propose a distinctive model wherein RecQ melts dsDNA internally toinitiate unwinding, and subsequently unwinds DNA as a dynamic assembly ofproteins cooperating at the fork.24-SubgKinesin-14: A League of their OwnSusan P. Gilbert.Rensselaer Polytechnic Institute, Troy, NY, USA.Kinesin-14 represents a subfamily of kinesins that are nonprocessive, promotemicrotubule (MT) minus-end-directed force generation, and contain C-terminalmotor domains that are dimerized through an N-terminal coiled-coil. Unlikethe well-known N-terminal motor domain kinesins that use an asymmetrichand-over-hand mechanism for MT plus-end-directed processive stepping,Kinesin-14s use a MT minus-end-directed powerstroke to generate force tocrosslink and slide one MT relative to another. While most Kinesin-14s arehomodimers like Drosophila Ncd, S. cerevisiae Kar3Vik1 is a heterodimer.The C-terminal domain of Vik1 exhibits the structural fold of a kinesin motordomain, binds MTs independent of Kar3, yet lacks a nucleotide-binding site.Furthermore, Kar3Vik1 binds across adjacent MT protofilaments, a non-canonical MT binding configuration. The results indicate that Kar3Vik1 col-lides with the MT through Vik1, promoting MT binding by Kar3. The tightbinding of Kar3 destabilizes the Vik1 interaction with the MT, positioningKar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is asso-ciated with the rotation of the coiled-coil stalk, and post-powerstroke ATPhydrolysis is independent of Vik1 providing additional evidence that Vik1rotates with the coiled-coil during the powerstroke. Detachment of Kar3Vik1from the MT completes the cycle and allows the motor to return to its initialconformation. Supported by NIH GM54141.Subgroup: Nanoscale Biophysics25-SubgSingle Molecule EnzymologyJulio Fernandez.Columbia University, NY, USA.We use single molecule force-clamp techniques to study the activity and chem-ical mechanisms of oxidases and oxidoreductase enzymes. In this approach thelength of an extending protein is measured while the pulling force is activelykept constant with a feedback loop. Using the force-clamp technique we haveinvestigated the force-dependency of protein folding, unfolding and of chemicalreactions. For example, using various types of force pulses we can drive a singlesubstrate protein to well defined extended states and monitor the reduction of itsdisulfide bonds by thioredoxin enzymes, with sub-Angstrom resolution (1). Byvarying the pulling force on the substrate we can also identify different chemicalmechanisms of reduction. Using maximum likelihood techniques we haveresuscitated ancient thioredoxin enzymes, going back billions of years. Wehave studied the activity of our resuscitated enzymes using our single moleculetechniques in an effort to observe the evolution of the chemical mechanisms ofreduction over time(2). Finally, we have used these single molecule techniquesto study the activity of oxidases such as PDI and DsbA, and of glutharedoxin(3).Our results demonstrate that single molecule force-spectroscopy techniquesprovide an entirely novel and powerful approach to study enzymes, at an un-precedented resolution. These experiments show that force-clamp AFM probesdynamic rearrangements within an enzyme’s active site, which cannot be re-solved by any other current structural biological technique. We anticipate thatthese studies will be extended to a wide range of other enzymes.1.Wiita et al, (2007) Probing the chemistry of thioredoxin catalysis with force.Nature, 450:124-7.2.Perez-Jimenez, et al, (2011). Single-molecule paleoenzymology probes thechemistry of resurrected enzymes. Nat Struct Mol Biol, 18(5): 592-596.3.Kosuri, P et al, (2012). Protein folding drives disulfide formation. Cell, inpress26-SubgDynamics of the Ribosomal Subunit Interface during TRNA Translocationat Near-Atomic ResolutionHelmut Grubmuller, PD Dr.Theoretical and Computational Biophysics Department, Max Planck Institutefor Biophysical Chemistry, Goettingen, Germany.During protein synthesis, movement of the transfer RNAs during translocationinvolves large structural motions of both the ribosome and the tRNAs. In par-ticular, large scale collective inter-subunit rotations occur. Despite this rotation,the two ribosomal subunits remain bound during translocation via contactpatches on the surface of the subunits. Combining crystal and cryo-EM struc-tures with all-atom explicit solvent molecular dynamics simulations, we havecharacterised intermediate states and conformational motions of spontaneoustRNA translocation at near-atomic resolution. Systematic analysis of the dy-namics of all inter-subunit contact patches suggest mechanisms by which thesubunits communicate, and how the two subunits maintain their fine-tunedSaturday, February 2, 2013 5aaffinity despite drastic conformational changes. The results are important forunderstanding the fundamental principles and underlying forces that generatemovement within molecular machines.27-SubgNew Insight into Lipid-Protein Membrane Organization and its Function-ality with Super-Resolution STED MicroscopyChristian Eggeling1,2, Veronica Mueller3, Alf Honigmann3,Debora M. Andrade3, Jorge Bernardino de la Serna2, Stefan W. Hell3.1Dept Nanobiophotonics, Max Planck Institute for biophysical Chemistry,Goettingen, Germany, 2Weatherall Institute of Molecular Medicine,University of Oxford, United Kingdom, 3Max Planck Institute for biophysicalChemistry, Goettingen, Germany.Stimulated Emission Depletion (STED) far-field microscopy allows the studyof living cells with nanoscale resolution, otherwise impeded by the limited spa-tial resolution of conventional microscopes. Besides the recording of images,the combination of STED with single-molecule sensitive spectroscopic toolssuch as Fluorescence Correlation Spectroscopy (FCS) discloses complexdynamical processes hidden to the conventional observations. For example,STED-FCS offers novel insights into important cellular processes, such aslipid-lipid, lipid-protein interactions or the formation of so-called ‘‘lipid-rafts’’in the cellular plasma membrane, and their role in cellular functionality.Improved insights are realized by the implementation of gated detection orby recording STED-FCS data during scanning.28-SubgProteins as Mechano-Chemical Signalling SwitchesViola Vogel, Prof. Dr.Department of Materials heading the Laboratory for Biologically OrientedMaterials, ETH Zurich, Zurich, Switzerland.Since the physical and biochemical properties of extracellular matrix providecritical cues to bacteria and cells, from mechanoregulated bacterial adhesionto angiogenesis, and finally to the differentiation of stem cells, it is of majorimportance to gain mechanistic insights into how mechanical stretching of ex-tracellular matrix molecules can alter various cell functions. While investigat-ing these three distinct physiological processes, common motifs are emerginghow bacteria and cells take advantage of mechanical forces to regulate the func-tion of proteins by stretching them out of their equilibrium structures. In thiscontext, new assays and techniques were developed that allow probing howthe stretching of proteins alters their structure-function relationships. Taken to-gether, new insights into various underpinning mechanotransduction events areemerging how mechanical cues are translated into biochemical signals thatultimately regulate bacterial adhesion and various cellular processes.Subgroup: Intrinsically Disordered Proteins29-SubgStructural Studies on the Activation and Substrate Binding of a Condition-ally Disordered Acid-Activated ChaperoneLinda Foit1, Bin Zhang2, Jenny George1, Lucia Brunetti2, Charles Brooks III2,James C.A. Bardwell1.1University of Michigan, Howard Hughes Medical Institute, MI, USA,2University of Michigan, Department of Chemistry and Biophysics Program,MI, USA.HdeA is a 9.7kDa chaperone that is inactive at neutral pH but becomes activatedand disordered by shifts to low pH. The chaperone is required for bacterial path-ogens to resist the acid-mediated protein aggregation that would otherwise occurin the human gut.We sought to identifywhich of HdeA’s acid-titratable residueswere the key players in its activation. Using constant pH molecular dynamicscalculations and site-specific mutagenesis, we identified several residues in-volved in HdeA activation and have isolated variants that are destabilized andconstitutively active at even neutral pH. These mutants help us to understandhow pH-driven changes in HdeA flexibility drive activation.One of the pressing problems in chaperone biology is how chaperones interactwith a multitude of client proteins to facilitate their folding. HdeA’s size andaccessibility to NMR also makes it ideal for monitoring the structural changesthat take place in this chaperone upon activation and upon client binding. Ourpredicted NMR structure of HdeA at neutral pH is a folded protein and verysimilar the crystal structures previously solved for the HdeA dimer. Upon shiftto low pH however, HdeA simultaneously becomes active as a chaperone andacquires a largely disordered conformation. We have obtained structural infor-mation that gives us insights into HdeA’s activation process and client binding.The use of small NMR accessible client proteins raises the exciting possibilityof monitoring the structural changes that take place within the client upon in-teraction with the chaperone and in doing so gaining insight into the fundamen-tal question of what chaperones do to their clients to facilitate folding.30-SubgAlpha-Synuclein, an Intrinsically Unstructured Protein. How InterestingCan It Be?Alexander Maltsev, Jinfa Ying, Yang Shen, Ad Bax.National Institutes of Health, MD, USA.N-terminal acetylation of alpha-synuclein (aS), a 140-residue protein impli-cated in the etiology of Parkinson’s disease, is common in mammals. Theimpact of this modification on the protein’s structure and dynamics in free so-lution and on its membrane binding properties has been evaluated by bothNMR and CD spectroscopy. While in contrast to literature reports, no tetra-meric form of acetylated aS could be isolated, N-terminal acetylation resultedin ca 15% transient population of alpha-helical structure for its first six residues.The 1H, 15N, and 13C chemical shifts for residues 13-140 remain unaffectedby acetylation. Nevertheless, a substantial increase in affinity of aS for nega-tively charged lipid membranes is observed, likely to be of strong functionalsignificance. A new method for residue-specific NMR probing of lipid bindingis demonstrated for aS and assigns a new putative function to this enigmaticprotein. Although free aS in the absence of lipids has backbone chemical shiftsthat are exceptionally close to random coil values, considerable positional var-iation for the distribution of its backbone torsion angles and the time scale oflocal reorientation can be deduced from its 3JHH and NOE data.31-SubgIntrinsic Protein Disorder in the Regulation of Large Molecular MachinesElisar Barbar, PhD.Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.We here report the molecular overlap of the linkage of three essential proteincomplexes that coordinate the formation of the mitotic spindle. These proteinsare dynein, a large motor complex that moves machinery inside cells, and twoof its regulators: a protein complex called dynactin, that is a dynein activator,and a protein called NudE whose depletion in mice produces a small brain andmental retardation. What is intriguing about the dynein/dynactin/NudE inter-play is that dynactin and NudE bind to a common segment of dynein that isintrinsically disordered but with distinct binding modes. Elucidating details ofthese distinct modes explains how one regulator is selected over the othereven when both are present in the same cellular compartment. UsingNMR spec-troscopy and isothermal titration calorimetry we show that intrinsic disorder ina specific segment of dynein intermediate chain promotes local modificationslike phosphorylation and splicing, promotes rapid equilibration of ensemblecomponents in solution samples of complexes with moderate to weak bindingaffinities, and creates a bi-segmental binding site such that residue-level modifi-cation in and near one segment haveminimal effect on the structure of the secondsegment thereby aiding in segregatingof functions between twoconsecutive seg-ments. These results underscore the role of disorder in the versatility of dyneinbinding to different regulators, and have far reaching impact not only on our un-derstanding of processes essential for formation and orientation of the spindle,but also offer a novel role for protein disorder in controlling cellular processes,and highlight the advantages of NMR spectroscopy in elucidating atomic levelcharacterization of extremely complex dynamic cellular assemblies.32-SubgProbing the Polymeric Properties of Unfolded and Disordered Proteinswith Single-Molecule SpectroscopyBenjamin Schuler.University of Zurich, Zurich, Switzerland.Single-molecule spectroscopy provides new opportunities for investigating thestructure and dynamics of unfolded and intrinsically disordered proteins(IDPs). The combination of single-molecule Förster resonance energy transfer(FRET) with nanosecond correlation spectroscopy, microfluidic mixing, andrelated methods can be used to probe their distance distributions and reconfigu-ration dynamics on a wide range of time scales, and even in heterogeneousenvironments. In view of the large structural heterogeneity of these systems,a description in terms of polymer physical principles is often a usefulway of con-ceptualizing their behavior. I will provide examples ranging from the influenceof amino acid composition and temperature on the structure and dynamics of un-folded proteins and IDPs to the effects of crowding and molecular chaperones.33-SubgA Long Disordered Linker in Nuclear Transport of Membrane ProteinsLiesbeth M. Veenhoff.University Medical Centre Groningen, University of Groningen, Groningen,Netherlands.Nuclear Pore Complexes (NPCs) embedded in the nuclear envelope allow se-lective transport of macromolecules between the cytosol and nucleoplasm.Transport factors shuttle cargo though the NPCs by interacting with the disor-dered proteins that encode the binding sites for the transport factors, the

English

4a Saturday, February 2, 2013to actin, whereas fast myosins such as muscle myosin-2 and some class-1
myosin members have a low duty ratio (< 0.1). The members of the respective
groups are assumed to use different ways to couple conformational
changes at the nucleotide binding regions to changes that occur at the actin
binding sites. Conserved active-site elements termed switch-1 and switch-2
play a major role in the coupling mechanism; however, whether and to which
extent small variations in the sequence of switch-1 and switch-2 affect the duty
ratio of a given myosin, thus determining its ability for processive movement,
fast contractility or tension bearing remains unresolved. Based on structural
considerations and confirmed by mutational analyses, we identified key
residues in the nucleotide binding pocket that are responsible for making
ADP dissociation kinetics dependent on the concentration of free magnesium
ions. The exchange of a single amino acid in switch-2 affected the motor prop-
erties of all myosins tested, but also transformed low duty ratio motors into high
duty ratio and vice versa. In addition, x-ray structural analyses and molecular
modeling allowed us to relate the observed changes to altered coupling between
the active sites and actin binding regions. These results, together with our cell
biological studies demonstrating for the first time that magnesium ions have
a regulatory role on motor protein function in vivo reveal that, switch-2 can
act as magnesium sensor critically determining the mechanochemical proper-
ties of myosins.
22-Subg
From Single Molecule Fluctuation to Muscle Contraction: A Brownian
Model of A.F. Huxley’s Hypotheses
Toshio Yanagida1,2, Lorenzo Marcucci1, Mitsuhiro Iwaki2.
1University of Osaka, Japan, 2Riken QBiC, Osaka, Japan.
Adaptive force generation of muscle in response to external stimuli is the result
of thermally fluctuating, cyclical interactions between myosin and actin, which
together form the actomyosin complex. Normally, these fluctuations are mod-
elled using transition rate functions that are based on muscle fiber behaviour, in
a phenomenological fashion (Huxley , 1957; Huxley & Simmons, 1971). How-
ever, such a basis reduces the predictive power of these models. As an alterna-
tive, we propose a model which uses direct single molecule observations of
actomyosin fluctuations (Kitamura, et al. Nature 1999, BIOPHYSICS, 2005;
Iwaki et al. Nat. Chem Biol. 2009). We precisely estimate the actomyosin
potential bias and use diffusion theory to obtain a Brownian ratchet model
that reproduces the complete cross-bridge cycle. The model is validated by
simulating several macroscopic experimental conditions, while its interpreta-
tion is compatible with two different force-generating scenarios (Lorenzo &
Yanagida, PloS One, 2012).
23-Subg
Watching Individual DNA Helicases and Motor Proteins Behaving and
Misbehaving
Stephen Kowalczykowski.
Microbiology, University of California, Davis, Davis, CA, USA.
We can now watch individual proteins acting on single molecules of DNA.
Using these approaches, we have been imaging the translocation of motor pro-
teins, such as the ssDNA translocases and DNA helicases, RecBCD, RecQ, and
Sgs1, and the dsDNA translocases and chromatin-remodeling enzymes, Rad54
and Tid1 proteins.
The RecBCD enzyme comprises two motor subunits: RecB, a 3’/5’ SF1-
helicase, and RecD, a 5’/3’ SF1-helicase. Although the mean unwinding
rate of individual RecBCD enzyme molecules is close to ensemble measure-
ments, the rates of individual enzyme molecules vary widely. Individual
RecBCD molecules unwind DNA at constant rates, suggesting static, not
dynamic, disorder. This apparent conformational heterogeneity is static on
the experimental time scale of DNA unwinding. We discovered that transiently
halting a single enzyme-DNA complex changes the rates of the RecBCD mol-
ecule. We will demonstrate that the behavior of individual RBCD enzymes
manifests a basic tenet of the ergodic hypothesis, suggesting that ligand binding
kinetically traps a single conformer that is capable of redistributing to all other
states within the population of molecules at equilibrium.
Using TIRF microscopy, the helicase activity of RecQ was visualized on single
molecules of DNA using a fluorescent ssDNA sensor. By monitoring the for-
mation and progression of individual unwinding forks, we observe that both
the frequency of initiation and rate of unwinding are highly dependent on
RecQ concentration. We establish that unwinding forks can initiate internally
by melting into dsDNA and can proceed in both directions. The findings sug-
gest that initiation requires a RecQ dimer, and continued unwinding involves
the repeated cooperative action of multiple monomers at the DNA unwinding
fork. We propose a distinctive model wherein RecQ melts dsDNA internally to
initiate unwinding, and subsequently unwinds DNA as a dynamic assembly of
proteins cooperating at the fork.24-Subg
Kinesin-14: A League of their Own
Susan P. Gilbert.
Rensselaer Polytechnic Institute, Troy, NY, USA.
Kinesin-14 represents a subfamily of kinesins that are nonprocessive, promote
microtubule (MT) minus-end-directed force generation, and contain C-terminal
motor domains that are dimerized through an N-terminal coiled-coil. Unlike
the well-known N-terminal motor domain kinesins that use an asymmetric
hand-over-hand mechanism for MT plus-end-directed processive stepping,
Kinesin-14s use a MT minus-end-directed powerstroke to generate force to
crosslink and slide one MT relative to another. While most Kinesin-14s are
homodimers like Drosophila Ncd, S. cerevisiae Kar3Vik1 is a heterodimer.
The C-terminal domain of Vik1 exhibits the structural fold of a kinesin motor
domain, binds MTs independent of Kar3, yet lacks a nucleotide-binding site.
Furthermore, Kar3Vik1 binds across adjacent MT protofilaments, a non-
canonical MT binding configuration. The results indicate that Kar3Vik1 col-
lides with the MT through Vik1, promoting MT binding by Kar3. The tight
binding of Kar3 destabilizes the Vik1 interaction with the MT, positioning
Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is asso-
ciated with the rotation of the coiled-coil stalk, and post-powerstroke ATP
hydrolysis is independent of Vik1 providing additional evidence that Vik1
rotates with the coiled-coil during the powerstroke. Detachment of Kar3Vik1
from the MT completes the cycle and allows the motor to return to its initial
conformation. Supported by NIH GM54141.
Subgroup: Nanoscale Biophysics
25-Subg
Single Molecule Enzymology
Julio Fernandez.
Columbia University, NY, USA.
We use single molecule force-clamp techniques to study the activity and chem-
ical mechanisms of oxidases and oxidoreductase enzymes. In this approach the
length of an extending protein is measured while the pulling force is actively
kept constant with a feedback loop. Using the force-clamp technique we have
investigated the force-dependency of protein folding, unfolding and of chemical
reactions. For example, using various types of force pulses we can drive a single
substrate protein to well defined extended states and monitor the reduction of its
disulfide bonds by thioredoxin enzymes, with sub-Angstrom resolution (1). By
varying the pulling force on the substrate we can also identify different chemical
mechanisms of reduction. Using maximum likelihood techniques we have
resuscitated ancient thioredoxin enzymes, going back billions of years. We
have studied the activity of our resuscitated enzymes using our single molecule
techniques in an effort to observe the evolution of the chemical mechanisms of
reduction over time(2). Finally, we have used these single molecule techniques
to study the activity of oxidases such as PDI and DsbA, and of glutharedoxin(3).
Our results demonstrate that single molecule force-spectroscopy techniques
provide an entirely novel and powerful approach to study enzymes, at an un-
precedented resolution. These experiments show that force-clamp AFM probes
dynamic rearrangements within an enzyme’s active site, which cannot be re-
solved by any other current structural biological technique. We anticipate that
these studies will be extended to a wide range of other enzymes.
1.Wiita et al, (2007) Probing the chemistry of thioredoxin catalysis with force.
Nature, 450:124-7.
2.Perez-Jimenez, et al, (2011). Single-molecule paleoenzymology probes the
chemistry of resurrected enzymes. Nat Struct Mol Biol, 18(5): 592-596.
3.Kosuri, P et al, (2012). Protein folding drives disulfide formation. Cell, in
press
26-Subg
Dynamics of the Ribosomal Subunit Interface during TRNA Translocation
at Near-Atomic Resolution
Helmut Grubmuller, PD Dr.
Theoretical and Computational Biophysics Department, Max Planck Institute
for Biophysical Chemistry, Goettingen, Germany.
During protein synthesis, movement of the transfer RNAs during translocation
involves large structural motions of both the ribosome and the tRNAs. In par-
ticular, large scale collective inter-subunit rotations occur. Despite this rotation,
the two ribosomal subunits remain bound during translocation via contact
patches on the surface of the subunits. Combining crystal and cryo-EM struc-
tures with all-atom explicit solvent molecular dynamics simulations, we have
characterised intermediate states and conformational motions of spontaneous
tRNA translocation at near-atomic resolution. Systematic analysis of the dy-
namics of all inter-subunit contact patches suggest mechanisms by which the
subunits communicate, and how the two subunits maintain their fine-tuned
Saturday, February 2, 2013 5aaffinity despite drastic conformational changes. The results are important for
understanding the fundamental principles and underlying forces that generate
movement within molecular machines.
27-Subg
New Insight into Lipid-Protein Membrane Organization and its Function-
ality with Super-Resolution STED Microscopy
Christian Eggeling1,2, Veronica Mueller3, Alf Honigmann3,
Debora M. Andrade3, Jorge Bernardino de la Serna2, Stefan W. Hell3.
1Dept Nanobiophotonics, Max Planck Institute for biophysical Chemistry,
Goettingen, Germany, 2Weatherall Institute of Molecular Medicine,
University of Oxford, United Kingdom, 3Max Planck Institute for biophysical
Chemistry, Goettingen, Germany.
Stimulated Emission Depletion (STED) far-field microscopy allows the study
of living cells with nanoscale resolution, otherwise impeded by the limited spa-
tial resolution of conventional microscopes. Besides the recording of images,
the combination of STED with single-molecule sensitive spectroscopic tools
such as Fluorescence Correlation Spectroscopy (FCS) discloses complex
dynamical processes hidden to the conventional observations. For example,
STED-FCS offers novel insights into important cellular processes, such as
lipid-lipid, lipid-protein interactions or the formation of so-called ‘‘lipid-rafts’’
in the cellular plasma membrane, and their role in cellular functionality.
Improved insights are realized by the implementation of gated detection or
by recording STED-FCS data during scanning.
28-Subg
Proteins as Mechano-Chemical Signalling Switches
Viola Vogel, Prof. Dr.
Department of Materials heading the Laboratory for Biologically Oriented
Materials, ETH Zurich, Zurich, Switzerland.
Since the physical and biochemical properties of extracellular matrix provide
critical cues to bacteria and cells, from mechanoregulated bacterial adhesion
to angiogenesis, and finally to the differentiation of stem cells, it is of major
importance to gain mechanistic insights into how mechanical stretching of ex-
tracellular matrix molecules can alter various cell functions. While investigat-
ing these three distinct physiological processes, common motifs are emerging
how bacteria and cells take advantage of mechanical forces to regulate the func-
tion of proteins by stretching them out of their equilibrium structures. In this
context, new assays and techniques were developed that allow probing how
the stretching of proteins alters their structure-function relationships. Taken to-
gether, new insights into various underpinning mechanotransduction events are
emerging how mechanical cues are translated into biochemical signals that
ultimately regulate bacterial adhesion and various cellular processes.
Subgroup: Intrinsically Disordered Proteins
29-Subg
Structural Studies on the Activation and Substrate Binding of a Condition-
ally Disordered Acid-Activated Chaperone
Linda Foit1, Bin Zhang2, Jenny George1, Lucia Brunetti2, Charles Brooks III2,
James C.A. Bardwell1.
1University of Michigan, Howard Hughes Medical Institute, MI, USA,
2University of Michigan, Department of Chemistry and Biophysics Program,
MI, USA.
HdeA is a 9.7kDa chaperone that is inactive at neutral pH but becomes activated
and disordered by shifts to low pH. The chaperone is required for bacterial path-
ogens to resist the acid-mediated protein aggregation that would otherwise occur
in the human gut.We sought to identifywhich of HdeA’s acid-titratable residues
were the key players in its activation. Using constant pH molecular dynamics
calculations and site-specific mutagenesis, we identified several residues in-
volved in HdeA activation and have isolated variants that are destabilized and
constitutively active at even neutral pH. These mutants help us to understand
how pH-driven changes in HdeA flexibility drive activation.
One of the pressing problems in chaperone biology is how chaperones interact
with a multitude of client proteins to facilitate their folding. HdeA’s size and
accessibility to NMR also makes it ideal for monitoring the structural changes
that take place in this chaperone upon activation and upon client binding. Our
predicted NMR structure of HdeA at neutral pH is a folded protein and very
similar the crystal structures previously solved for the HdeA dimer. Upon shift
to low pH however, HdeA simultaneously becomes active as a chaperone and
acquires a largely disordered conformation. We have obtained structural infor-
mation that gives us insights into HdeA’s activation process and client binding.
The use of small NMR accessible client proteins raises the exciting possibility
of monitoring the structural changes that take place within the client upon in-
teraction with the chaperone and in doing so gaining insight into the fundamen-
tal question of what chaperones do to their clients to facilitate folding.30-Subg
Alpha-Synuclein, an Intrinsically Unstructured Protein. How Interesting
Can It Be?
Alexander Maltsev, Jinfa Ying, Yang Shen, Ad Bax.
National Institutes of Health, MD, USA.
N-terminal acetylation of alpha-synuclein (aS), a 140-residue protein impli-
cated in the etiology of Parkinson’s disease, is common in mammals. The
impact of this modification on the protein’s structure and dynamics in free so-
lution and on its membrane binding properties has been evaluated by both
NMR and CD spectroscopy. While in contrast to literature reports, no tetra-
meric form of acetylated aS could be isolated, N-terminal acetylation resulted
in ca 15% transient population of alpha-helical structure for its first six residues.
The 1H, 15N, and 13C chemical shifts for residues 13-140 remain unaffected
by acetylation. Nevertheless, a substantial increase in affinity of aS for nega-
tively charged lipid membranes is observed, likely to be of strong functional
significance. A new method for residue-specific NMR probing of lipid binding
is demonstrated for aS and assigns a new putative function to this enigmatic
protein. Although free aS in the absence of lipids has backbone chemical shifts
that are exceptionally close to random coil values, considerable positional var-
iation for the distribution of its backbone torsion angles and the time scale of
local reorientation can be deduced from its 3JHH and NOE data.
31-Subg
Intrinsic Protein Disorder in the Regulation of Large Molecular Machines
Elisar Barbar, PhD.
Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.
We here report the molecular overlap of the linkage of three essential protein
complexes that coordinate the formation of the mitotic spindle. These proteins
are dynein, a large motor complex that moves machinery inside cells, and two
of its regulators: a protein complex called dynactin, that is a dynein activator,
and a protein called NudE whose depletion in mice produces a small brain and
mental retardation. What is intriguing about the dynein/dynactin/NudE inter-
play is that dynactin and NudE bind to a common segment of dynein that is
intrinsically disordered but with distinct binding modes. Elucidating details of
these distinct modes explains how one regulator is selected over the other
even when both are present in the same cellular compartment. UsingNMR spec-
troscopy and isothermal titration calorimetry we show that intrinsic disorder in
a specific segment of dynein intermediate chain promotes local modifications
like phosphorylation and splicing, promotes rapid equilibration of ensemble
components in solution samples of complexes with moderate to weak binding
affinities, and creates a bi-segmental binding site such that residue-level modifi-
cation in and near one segment haveminimal effect on the structure of the second
segment thereby aiding in segregatingof functions between twoconsecutive seg-
ments. These results underscore the role of disorder in the versatility of dynein
binding to different regulators, and have far reaching impact not only on our un-
derstanding of processes essential for formation and orientation of the spindle,
but also offer a novel role for protein disorder in controlling cellular processes,
and highlight the advantages of NMR spectroscopy in elucidating atomic level
characterization of extremely complex dynamic cellular assemblies.
32-Subg
Probing the Polymeric Properties of Unfolded and Disordered Proteins
with Single-Molecule Spectroscopy
Benjamin Schuler.
University of Zurich, Zurich, Switzerland.
Single-molecule spectroscopy provides new opportunities for investigating the
structure and dynamics of unfolded and intrinsically disordered proteins
(IDPs). The combination of single-molecule Fo¨rster resonance energy transfer
(FRET) with nanosecond correlation spectroscopy, microfluidic mixing, and
related methods can be used to probe their distance distributions and reconfigu-
ration dynamics on a wide range of time scales, and even in heterogeneous
environments. In view of the large structural heterogeneity of these systems,
a description in terms of polymer physical principles is often a usefulway of con-
ceptualizing their behavior. I will provide examples ranging from the influence
of amino acid composition and temperature on the structure and dynamics of un-
folded proteins and IDPs to the effects of crowding and molecular chaperones.
33-Subg
A Long Disordered Linker in Nuclear Transport of Membrane Proteins
Liesbeth M. Veenhoff.
University Medical Centre Groningen, University of Groningen, Groningen,
Netherlands.
Nuclear Pore Complexes (NPCs) embedded in the nuclear envelope allow se-
lective transport of macromolecules between the cytosol and nucleoplasm.
Transport factors shuttle cargo though the NPCs by interacting with the disor-
dered proteins that encode the binding sites for the transport factors, the


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Dynamics of the Ribosomal Subunit Interface during TRNA Translocation at Near-Atomic Resolution

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