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Bert L de Groot

Publications and source records attributed to Bert L de Groot.

16 recordsLinked to original sources
PubMed2006

Does CO2 permeate through aquaporin-1?

Aquaporins facilitate water permeation across biological membranes. Additionally, glycerol and other small neutral solutes are permeated by related aquaglyceroporins. The role of aquaporins in gas permeation has been a long-standing and controversially discussed issue. We present an extensive set of atomistic molecular dynamics simulations that address the question of CO(2) permeation through human aquaporin-1. Free energy profiles derived from the simulations display a barrier of approximately 23 kJ/mol in the aromatic/arginine constriction region of the water pore, whereas a barrier of approximately 4 kJ/mol was observed for a palmitoyloleoylphosphatidylethanolamine lipid bilayer membrane. The results indicate that significant aquaporin-1-mediated CO(2) permeation is to be expected only in membranes with a low intrinsic CO(2) permeability.

Aquaporin 1↗
PubMed2006

Mobility of a one-dimensional confined file of water molecules as a function of file length.

Confinement of water by pore geometry to a one-dimensional file of molecules interacting with the pore alters the diffusion coefficient D(W). Here we report an exponential dependence of D(W) on the number of water positions in the pore. The result is based on measurements of single channel water permeabilities of structurally similar peptidic nanopores of different length. The inconsistency with predictions from continuum or kinetic models indicates that pore occupancy is reduced in single file transport. In longer pores (e.g., in aquaporins) the presence of charged residues increases D(W).

Biological Transport↗
PubMed2006

Quaternary ammonium compounds as water channel blockers. Specificity, potency, and site of action.

Excessive water uptake through Aquaporins (AQP) can be life-threatening and reversible AQP inhibitors are needed. Here, we determined the specificity, potency, and binding site of tetraethylammonium (TEA) to block Aquaporin water permeability. Using oocytes, externally applied TEA blocked AQP1/AQP2/AQP4 with IC50 values of 1.4, 6.2, and 9.8 microM, respectively. Related tetraammonium compounds yielded some (propyl) or no (methyl, butyl, or pentyl) inhibition. TEA inhibition was lost upon a Tyr to Phe amino acid switch in the external water pore of AQP1/AQP2/AQP4, whereas the water permeability of AQP3 and AQP5, which lack a corresponding Tyr, was not blocked by TEA. Consistent with experimental data, multi-nanosecond molecular dynamics simulations showed one stable binding site for TEA, but not tetramethyl (TMA), in AQP1, resulting in a nearly 50% water permeability inhibition, which was reduced in AQP1-Y186F due to effects on the TEA inhibitory binding region. Moreover, in the simulation TEA interacted with charged residues in the C (Asp128) and E (Asp185) loop, and the A(Tyr37-Asn42-Thr44) loop of the neighboring monomer, but not directly with Tyr186. The loss of TEA inhibition in oocytes expressing properly folded AQP1-N42A or -T44A is in line with the computationally predicted binding mode. Our data reveal that the molecular interaction of TEA with AQP1 differs and is about 1000-fold more effective on AQPs than on potassium channels. Moreover, the observed experimental and simulated similarities open the way for rational design and virtual screening for AQP-specific inhibitors, with quaternary ammonium compounds in general, and TEA in particular as a lead compound.

Amino Acid Sequence↗
PubMed2006

Alternate structural conformations of Streptococcus pneumoniae hyaluronan lyase: insights into enzyme flexibility and underlying molecular mechanism of action.

Streptococcus pneumoniae hyaluronan lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades several biologically important, information-rich linear polymeric glycans: hyaluronan, unsulfated chondroitin, and some chondroitin sulfates. This degradation facilitates spreading of bacteria throughout the host tissues and presumably provides energy and a carbon source for pneumococcal cells. Its beta-elimination catalytic mechanism is an acid/base process termed proton acceptance and donation leading to cleavage of beta-1,4 linkages of the substrates. The degradation of hyaluronan occurs in two stages, initial endolytic cuts are followed by processive exolytic cleavage of one disaccharide at a time. In contrast, the degradation of chondroitins is purely endolytic. Structural studies together with flexibility analyses of two streptococcal enzymes, from S.pneumoniae and Streptococcus agalactiae, allowed for insights into this enzyme's molecular mechanism. Here, two new X-ray crystal structures of the pneumococcal enzyme in novel conformations are reported. These new conformations, complemented by molecular dynamics simulation results, directly confirm the predicted domain motions presumed to facilitate the processive degradative process. One of these new structures resembles the S.agalactiae enzyme conformation, and provides evidence of a uniform mechanistic/dynamic behavior of this protein across different bacteria.

Catalytic Domain↗
PubMed2006

Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles.

During exocytosis a four-helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP-25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C-terminal end affected fusion triggering in vivo and led to two-step unfolding of the SNARE complex in vitro, indicating that the C-terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C-terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N-terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N- to C-terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.

Amino Acid Sequence↗
PubMed2006

Ligand-release pathways in the pheromone-binding protein of Bombyx mori.

Pheromone-binding proteins (PBP) supply olfactory neuron cells with pheromones by binding the ligands they are tailored for and carrying them to their receptor. The function of a PBP as an efficient carrier requires fast ligand uptake and release. The molecular basis of the ligand-binding mechanism was addressed here for the intriguing case of the PBP of the silk moth Bombyx mori. This PBP completely encapsulates its ligand bombykol without displaying any obvious ligand entrance/exit sites. Here, two opposite dissociation routes were identified as the most likely entrance/exit paths by replica-exchange molecular dynamics, essential dynamics, and force-probe molecular dynamics simulations. One of the paths runs along a flexible front lid; the other along the termini at the back. Calculated forces and energies suggest that both routes are physiologically relevant. The multiplicity of pathways may reduce or tune the entropic barrier for ligand binding.

Animals↗
PubMed2005

Alternative splicing of SNAP-25 regulates secretion through nonconservative substitutions in the SNARE domain.

The essential membrane fusion apparatus in mammalian cells, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consists of four alpha-helices formed by three proteins: SNAP-25, syntaxin 1, and synaptobrevin 2. SNAP-25 contributes two helices to the complex and is targeted to the plasma membrane by palmitoylation of four cysteines in the linker region. It is alternatively spliced into two forms, SNAP-25a and SNAP-25b, differing by nine amino acids substitutions. When expressed in chromaffin cells from SNAP-25 null mice, the isoforms support different levels of secretion. Here, we investigated the basis of that different secretory phenotype. We found that two nonconservative substitutions in the N-terminal SNARE domain and not the different localization of one palmitoylated cysteine cause the functional difference between the isoforms. Biochemical and molecular dynamic simulation experiments revealed that the two substitutions do not regulate secretion by affecting the property of SNARE complex itself, but rather make the SNAP-25b-containing SNARE complex more available for the interaction with accessory factor(s).

Alternative Splicing↗
PubMed2005

The 4.5 A structure of human AQP2.

Located in the principal cells of the collecting duct, aquaporin-2 (AQP2) is responsible for the regulated water reabsorption in the kidney and is indispensable for the maintenance of body water balance. Disregulation or malfunctioning of AQP2 can lead to severe diseases such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. Here we present the crystallization of recombinantly expressed human AQP2 into two-dimensional protein-lipid arrays and their structural characterization by atomic force microscopy and electron crystallography. These crystals are double-layered sheets that have a diameter of up to 30 microm, diffract to 3 A(-1) and are stacked by contacts between their cytosolic surfaces. The structure determined to 4.5 A resolution in the plane of the membrane reveals the typical aquaporin fold but also a particular structure between the stacked layers that is likely to be related to the cytosolic N and C termini.

Aquaporin 2↗
PubMed2005

Probing conformational disorder in neurotensin by two-dimensional solid-state NMR and comparison to molecular dynamics simulations.

An approach is introduced to characterize conformational ensembles of intrinsically unstructured peptides on the atomic level using two-dimensional solid-state NMR data and their combination with molecular dynamics simulations. For neurotensin, a peptide that binds with high affinity to a G-protein coupled receptor, this method permits the investigation of the changes in conformational preferences of a neurotransmitter transferred from a frozen aqueous solution via a lipid model phase to the receptor-bound form. The results speak against a conformational pre-organization of the ligand in detergents in which the receptor has been shown to be functional. Further extensions to the study of protein folding are possible.

Detergents↗
PubMed2005

The dynamics and energetics of water permeation and proton exclusion in aquaporins.

Aquaporins and aquaglyceroporins are passive membrane channels that, in many species, facilitate highly efficient yet strictly selective permeation of water and small solutes across lipid bilayers. Their ability to block proton flux is particularly remarkable, because other aqueous pores and water efficiently conduct protons, via the so-called Grotthuss mechanism. How efficient water permeation is achieved and how it is reconciled with the seemingly contradictory task of strict proton exclusion have been long-standing puzzles. Because neither the dynamics of the water molecules nor the mobility of protons inside the aquaporin channel could be experimentally accessed so far, several groups addressed this challenge using a variety of atomistic computer simulation methods.

Aquaporins↗
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↗
PubMed2002

Mechanism of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. Structures of complexes with the substrate.

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniae hyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-A resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn(349), His(399), and Tyr(408), is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the alpha- and beta-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.

Amino Acid Sequence↗
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

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↗