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H Grubmüller

Publications and source records attributed to H Grubmüller.

12 recordsLinked to original sources
PubMed2003

Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1.

Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.

Amino Acid Substitution↗
PubMed2002

Progress in the analysis of membrane protein structure and function.

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.

Crystallization↗
PubMed2001

Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF.

"Real time" molecular dynamics simulations of water permeation through human aquaporin-1 (AQP1) and the bacterial glycerol facilitator GlpF are presented. We obtained time-resolved, atomic-resolution models of the permeation mechanism across these highly selective membrane channels. Both proteins act as two-stage filters: Conserved fingerprint [asparagine-proline-alanine (NPA)] motifs form a selectivity-determining region; a second (aromatic/arginine) region is proposed to function as a proton filter. Hydrophobic regions near the NPA motifs are rate-limiting water barriers. In AQP1, a fine-tuned water dipole rotation during passage is essential for water selectivity. In GlpF, a glycerol-mediated "induced fit" gating motion is proposed to generate selectivity for glycerol over water.

Amino Acid Motifs↗
PubMed2001

A refined structure of human aquaporin-1.

A refined structure of the human water channel aquaporin-1 is presented. The model rests on the high resolution X-ray structure of the homologous bacterial glycerol transporter GlpF, electron crystallographic data at 3.8 A resolution and a multiple sequence alignment of the aquaporin superfamily. The crystallographic R and free R values (36.7% and 37.8%) for the refined structure are significantly lower than for previous models. Improved geometry and enhanced stability in molecular dynamics simulations demonstrate a significant improvement of the aquaporin-1 structure. Comparison with previous aquaporin-1 models shows significant differences, not only in the loop regions, but also in the core of the water channel.

Aquaporin 1↗
PubMed2001

Essential dynamics of reversible peptide folding: memory-free conformational dynamics governed by internal hydrogen bonds.

A principal component analysis has been applied on equilibrium simulations of a beta-heptapeptide that shows reversible folding in a methanol solution. The analysis shows that the configurational space contains only three dense sub-states. These states of relatively low free energy correspond to the "native" left-handed helix, a partly helical intermediate, and a hairpin-like structure. The collection of unfolded conformations form a relatively diffuse cloud with little substructure. Internal hydrogen-bonding energies were found to correlate well with the degree of folding. The native helical structure folds from the N terminus; the transition from the major folding intermediate to the native helical structure involves the formation of the two most C-terminal backbone hydrogen bonds. A four-state Markov model was found to describe transition frequencies between the conformational states within error limits, indicating that memory-effects are negligible beyond the nanosecond time-scale. The dominant native state fluctuations were found to be very similar to unfolding motions, suggesting that unfolding pathways can be inferred from fluctuations in the native state. The low-dimensional essential subspace, describing 69% of the collective atomic fluctuations, was found to converge at time-scales of the order of one nanosecond at all temperatures investigated, whereas folding/unfolding takes place at significantly longer time-scales, even above the melting temperature.

Computer Simulation↗
PubMed2001

Molecular dynamics force probe simulations of antibody/antigen unbinding: entropic control and nonadditivity of unbinding forces.

Unbinding of a spin-labeled dinitrophenyl (DNP) hapten from the monoclonal antibody AN02 F(ab) fragment has been studied by force probe molecular dynamics (FPMD) simulations. In our nanosecond simulations, unbinding was enforced by pulling the hapten molecule out of the binding pocket. Detailed inspection of the FPMD trajectories revealed a large heterogeneity of enforced unbinding pathways and a correspondingly large flexibility of the binding pocket region, which exhibited induced fit motions. Principal component analyses were used to estimate the resulting entropic contribution of approximately 6 kcal/mol to the AN02/DNP-hapten bond. This large contribution may explain the surprisingly large effect on binding kinetics found for mutation sites that are not directly involved in binding. We propose that such "entropic control" optimizes the binding kinetics of antibodies. Additional FPMD simulations of two point mutants in the light chain, Y33F and I96K, provided further support for a large flexibility of the binding pocket. Unbinding forces were found to be unchanged for these two mutants. Structural analysis of the FPMD simulations suggests that, in contrast to free energies of unbinding, the effect of mutations on unbinding forces is generally nonadditive.

Antibodies, Monoclonal↗
PubMed2000

Exocytosis requires asymmetry in the central layer of the SNARE complex.

Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

Alleles↗
PubMed2000

The fold of human aquaporin 1.

The fold of human aquaporin 1 is determined from cryo-electron microscopic data at 4.5 A resolution. The monomeric structure consists of two transmembrane triple helices arranged around a pseudo-2-fold axis connected by a long flexible extracellular loop. Each triplet contains between its second and third helix a functional loop containing the highly conserved fingerprint NPA motif. These functional loops are assumed to fold inwards between the two triplets, thereby forming the heart of the water channel. The helix topology was determined from the directionality pattern of each of the six transmembrane helices with respect to the membrane, together with constraints defined by the sequence and atomic force microscopy data. The directionality of the helices was determined by collecting the best-fitting orientations resulting from a search through the three-dimensional experimental map for a large number of alpha-helical fragments. Tests on cryo-electron crystallographic bacteriorhodopsin data suggest that our method is generally applicable to determine the topology of helical proteins for which only medium-resolution electron microscopy data are available.

Aquaporin 1↗
PubMed2000

Dynamic force spectroscopy of molecular adhesion bonds.

Recent advances in atomic force microscopy, biomembrane force probe experiments, and optical tweezers allow one to measure the response of single molecules to mechanical stress with high precision. Such experiments, due to limited spatial resolution, typically access only one single force value in a continuous force profile that characterizes the molecular response along a reaction coordinate. We develop a theory that allows one to reconstruct force profiles from force spectra obtained from measurements at varying loading rates, without requiring increased resolution. We show that spectra obtained from measurements with different spring constants contain complementary information.

Journal Article↗
PubMed1996

Ligand binding: molecular mechanics calculation of the streptavidin-biotin rupture force.

The force required to rupture the streptavidin-biotin complex was calculated here by computer simulations. The computed force agrees well with that obtained by recent single molecule atomic force microscope experiments. These simulations suggest a detailed multiple-pathway rupture mechanism involving five major unbinding steps. Binding forces and specificity are attributed to a hydrogen bond network between the biotin ligand and residues within the binding pocket of streptavidin. During rupture, additional water bridges substantially enhance the stability of the complex and even dominate the binding interactions. In contrast, steric restraints do not appear to contribute to the binding forces, although conformational motions were observed.

Bacterial Proteins↗
PubMed1990

Self-organization of associative memory and pattern classification: recurrent signal processing on topological feature maps.

We extend the neural concepts of topological feature maps towards self-organization of auto-associative memory and hierarchical pattern classification. As is well-known, topological maps for statistical data sets store information on the associated probability densities. To extract that information we introduce a recurrent dynamics of signal processing. We show that the dynamics converts a topological map into an auto-associative memory for real-valued feature vectors which is capable to perform a cluster analysis. The neural network scheme thus developed represents a generalization of non-linear matrix-type associative memories. The results naturally lead to the concept of a feature atlas and an associated scheme of self-organized, hierarchical pattern classification.

Algorithms↗