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Helmut Grubmüller

Publications and source records attributed to Helmut Grubmüller.

33 records · Page 2Linked to original sources
PubMed2005

Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements.

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.

Computer Simulation↗
PubMed2004

Mechanically induced titin kinase activation studied by force-probe molecular dynamics simulations.

The conversion of mechanical stress into a biochemical signal in a muscle cell requires a force sensor. Titin kinase, the catalytic domain of the elastic muscle protein titin, has been suggested as a candidate. Its activation requires major conformational changes resulting in the exposure of its active site. Here, force-probe molecular dynamics simulations were used to obtain insight into the tension-induced activation mechanism. We find evidence for a sequential mechanically induced opening of the catalytic site without complete domain unfolding. Our results suggest the rupture of two terminal beta-sheets as the primary unfolding steps. The low force resistance of the C-terminal relative to the N-terminal beta-sheet is attributed to their different geometry. A subsequent rearrangement of the autoinhibitory tail is seen to lead to the exposure of the active site, as is required for titin kinase activity. These results support the hypothesis of titin kinase as a force sensor.

Computer Simulation↗
PubMed2004

Mechanical properties of single motor molecules studied by three-dimensional thermal force probing in optical tweezers.

A new method combining three-dimensional (3D) force measurements in an optical trap with the analysis of thermally induced (Brownian) position fluctuations of a trapped probe was used to investigate the mechanical properties of a single molecule, the molecular motor kinesin. One kinesin molecule attached to the probe was bound in a rigorlike state to one microtubule. The optical trap was kept weak to measure the thermal forces acting on the probe, which were mainly counterbalanced by the kinesin tether. The stiffness of kinesin during stretching and compression with respect to its backbone axis were measured. Our results indicate that a section of kinesin close to the motor domain is the dominating element in the flexibility of the motor structure. The experiments demonstrate the power of 3D thermal fluctuation analysis to characterize mechanical properties of individual motor proteins and indicate its usefulness to study single molecule in general

Journal Article↗
PubMed2004

Differential peptide dynamics is linked to major histocompatibility complex polymorphism.

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.

Entropy↗
PubMed2003

The mechanism of proton exclusion in the aquaporin-1 water channel.

Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity.

Animals↗
PubMed2003

The structure of the aquaporin-1 water channel: a comparison between cryo-electron microscopy and X-ray crystallography.

Three different medium-resolution structures of the human water channel aquaporin-1 (AQP1) have been solved by cryo-electron microscopy (cryo-EM) during the last two years. Recently, the structure of the strongly related bovine AQP1 was solved by X-ray crystallography at higher resolution, allowing a validation of the original medium-resolution structures, and providing a good indication for the strengths and limitations of state of the art cryo-EM methods. We present a detailed comparison between the different models, which shows that overall, the structures are highly similar, deviating less than 2.5 A from each other in the helical backbone regions. The two original cryo-EM structures, however, also show a number of significant deviations from the X-ray structure, both in the backbone positions of the transmembrane helices and in the location of the amino acid side-chains facing the pore. In contrast, the third cryo-EM structure that included information from the X-ray structure of the homologous bacterial glycerol facilitator GlpF and that was subsequently refined against cryo-EM AQP1 data, shows a root mean square deviation of 0.9A from the X-ray structure in the helical backbone regions.

Animals↗
PubMed2003

Conformational dynamics of the F1-ATPase beta-subunit: a molecular dynamics study.

According to the different nucleotide occupancies of the F(1)-ATPase beta-subunits and due to the asymmetry imposed through the central gamma-subunit, the beta-subunit adopts different conformations in the crystal structures. Recently, a spontaneous and nucleotide-independent closure of the open beta-subunit upon rotation of the gamma-subunit has been proposed. To address the question whether this closure is dictated by interactions to neighbored subunits or whether the open beta-subunit behaves like a prestressed "spring," we report multinanosecond molecular dynamics simulations of the isolated beta-subunit with different start conformations and different nucleotide occupancies. We have observed a fast, spontaneous closure motion of the open beta(E)-subunit, consistent with the available x-ray structures. The motions and kinetics are similar to those observed in simulations of the full (alpha beta)(3)gamma-complex, which support the view of a prestressed "spring," i.e., that forces internal to the beta(E)-subunit dominate possible interactions from adjacent alpha-subunits. Additionally, nucleotide removal is found to trigger conformational transitions of the closed beta(TP)-subunit; this provides evidence that the recently resolved half-closed beta-subunit conformation is an intermediate state before product release. The observed motions provide a plausible explanation why ADP and P(i) are required for the release of bound ATP and why gamma-depleted (alpha beta)(3) has a drastically reduced hydrolysis rate.

Adenosine Diphosphate↗
PubMed2003

Effect of sodium chloride on a lipid bilayer.

Electrostatic interactions govern structural and dynamical properties of membranes and can vary considerably with the composition of the aqueous buffer. We studied the influence of sodium chloride on a pure POPC lipid bilayer by fluorescence correlation spectroscopy experiments and molecular dynamics simulations. Increasing sodium chloride concentration was found to decrease the self-diffusion of POPC lipids within the bilayer. Self-diffusion coefficients calculated from the 100 ns simulations agree with those measured on a millisecond timescale, suggesting that most of the relaxation processes relevant for lipid diffusion are faster than the simulation timescale. As the dominant effect, the molecular dynamics simulations revealed a tight binding of sodium ions to the carbonyl oxygens of on average three lipids leading to larger complexes with reduced mobility. Additionally, the bilayer thickens by approximately 2 A, which increases the order parameter of the fatty acyl chains. Sodium binding alters the electrostatic potential, which is largely compensated by a changed polarization of the aqueous medium and a lipid dipole reorientation.

Biophysical Phenomena↗
PubMed2003

Mechanical coupling via the membrane fusion SNARE protein syntaxin 1A: a molecular dynamics study.

SNARE trans complexes between membranes likely promote membrane fusion. For the t-SNARE syntaxin 1A involved in synaptic transmission, the secondary structure and bending stiffness of the five-residue juxtamembrane linker is assumed to determine the required mechanical energy transfer from the cytosolic core complex to the membrane. These properties have here been studied by molecular dynamics and annealing simulations for the wild-type and a C-terminal-prolongated mutant within a neutral and an acidic bilayer, suggesting linker stiffnesses above 1.7 but below 50 x 10(-3) kcal mol(-1) deg(-2). The transmembrane helix was found to be tilted by 15 degrees and tightly anchored within the membrane with a stiffness of 4-5 kcal mol(-1) A(-2). The linker turned out to be marginally helical and strongly influenced by its lipid environment. Charged lipids increased the helicity and H3 helix tilt stiffness. For the wild type, the linker was seen embedded deeply within the polar region of the bilayer, whereas the prolongation shifted the linker outward. This reduced its helicity and increased its average tilt, thereby presumably reducing fusion efficiency. Our results suggest that partially unstructured linkers provide considerable mechanical coupling; the energy transduced cooperatively by the linkers in a native fusion event is thus estimated to be 3-8 kcal/mol, implying a two-to-five orders of magnitude fusion rate increase.

Antigens, Surface↗
PubMed2002

Force spectroscopy of single biomolecules.

Many processes in the body are effected and regulated by highly specialized protein molecules: These molecules certainly deserve the name "biochemical nanomachines". Recent progress in single-molecule experiments and corresponding simulations with supercomputers enable us to watch these "nanomachines" at work, revealing a host of astounding mechanisms. Examples are the fine-tuned movements of the binding pocket of a receptor protein locking into its ligand molecule and the forced unfolding of titin, which acts as a molecular shock absorber to protect muscle cells. At present, we are not capable of designing such high precision machines, but we are beginning to understand their working principles and to simulate and predict their function.

Ligands↗
PubMed2002

Water permeation through gramicidin A: desformylation and the double helix: a molecular dynamics study.

Multinanosecond molecular dynamics simulations of gramicidin A embedded in a dimyristoylphosphatidylcholine bilayer show a remarkable structural stability for both experimentally determined conformations: the head-to-head helical dimer and the double helix. Water permeability was found to be much higher in the double helical conformation, which is explained by lower hydrogen bond-mediated enthalpic barriers at the channel entrance and its larger pore size. Free-energy perturbation calculations show that the double helical structure is stabilized by the positive charges at the N termini introduced by the desformylation, whereas the helical dimer is destabilized. Together with the recent experimental observation that desformyl gramicidin conducts water hundredfold better than gramicidin, this suggests that desformyl gramicidin A predominantly occurs in the double helical conformation.

Biophysical Phenomena↗
PubMed2002

Membrane fusion.

Fusion of biological membranes is governed by physical principles but it is unclear whether the transition states are primarily determined by lipid physics or by protein-lipid interactions. Recent advances in the field include the physical description of bilayer fusion, particularly new models beyond continuum models and the role of the SNARE proteins. Despite substantial progress, an integrated concept for protein-mediated membrane fusion is not yet available, and many open questions remain to be answered.

Animals↗
PubMed2002

Structure and function of water channels.

All aquaporins are efficient water transporters, while sustaining strict selectivity, even against protons, thereby maintaining the proton gradient across the cell membrane. Recently solved structures of these membrane channels have helped us to understand this remarkable property.

Aquaporins↗
PubMed2002

Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F1-ATP synthase.

The mitochondrial membrane protein FoF1-ATP synthase synthesizes adenosine triphosphate (ATP), the universal currency of energy in the cell. This process involves mechanochemical energy transfer from a rotating asymmetric gamma-'stalk' to the three active sites of the F1 unit, which drives the bound ATP out of the binding pocket. Here, the primary structural changes associated with this energy transfer in F1-ATP synthase were studied with multi-nanosecond molecular dynamics simulations. By forced rotation of the gamma-stalk that mimics the effect of proton motive Fo-rotation during ATP synthesis, a time-resolved atomic model for the structural changes in the F1 part in terms of propagating conformational motions is obtained. For these, different time scales are found, which allows the separation of nanosecond from microsecond conformational motions. In the simulations, rotation of the gamma-stalk lowers the ATP affinity of the betaTP binding pocket and triggers fast, spontaneous closure of the empty betaE subunit. The simulations explain several mutation studies and the reduced hydrolysis rate of gamma-depleted F1-ATPase.

Adenosine Triphosphate↗