Small organic molecules, like Congo red and lacmoid, have been shown to modulate the self-assembly of the amyloid β peptide (Aβ). Here, we show that Aβ forms NMR invisible non-toxic co-aggregates together with lacmoid as well as Congo red. We find that the interaction involves two distinct kinetic processes and at every given time point only a small fraction of Aβ is in the co-aggregate. These weak transient interactions kinetically redirect the aggregation prone Aβ from self-assembling into amyloid fibrils. These findings suggest that even such weak binders might be effective as therapeutics against pathogenic protein aggregation.

The central theme in this thesis is reversibility. Two main attempts has been made to approach reversibility in cellular systems from both chemical and physical points of view. Reversibility of immunolabeling of proteins on the cell surface has been adressed by development of new fluorescent substances optimized for CALI (Chromophore-Assisted Laser Inactivation of protein). Aluminum phthalocyanine (AlPc) is here identified to be a good candidate for a new generation of fluorophores for efficient hydroxyl radical generation. It is shown that cells can be reversibly labeled with antibody-AlPc conjugates. In experiments on living cells the AlPcs were not only active as classic fluorophores but also as photocatalytic substances with destaining properties. Reversibility of cell immobilization is also reported, where cells cultured in microstructures were immobilized and 3D supported using hydrogels. Hydrogel formulation and application was optimized to achieve a system where both viability and ease of use was satisfied. Gel reversibility was actualized with pH and enzyme treatment. The developped method offers the possibility of stop flow culturing cells in controlled and reusable 3D environments.

Based on molecular dynamics simulations, an analysis of structure and dynamics is performed on interfacial water at a liquid crystalline dipalmitoylphosphatidycholine/water system. Water properties relevant for understanding NMR relaxation are emphasized. The first and second rank orientational order parameters of the water O-H bonds were calculated, where the second rank order parameter is in agreement with experimental determined quadrupolar splittings. Also, two different interfacial water regions (bound water regions) are revealed with respect to different signs of the second rank order parameter. The water reorientation correlation function reveals a mixture of fast and slow decaying parts. The fast (ps) part of the correlation function is due to local anisotropic water reorientation whereas the much slower part is due to more complicated processes including lateral diffusion along the interface and chemical exchange between free and bound water molecules. The 100-ns-long molecular dynamics simulation at constant pressure (1 atm) and at a temperature of 50 degrees C of 64 lipid molecules and 64 x 23 water molecules lack a slow water reorientation correlation component in the ns time scale. The (2)H(2)O powder spectrum of the dipalmitoylphosphatidycholine/water system is narrow and consequently, the NMR relaxation time T(2) is too short compared to experimental results.

The CPLEAR set-up (modified) has been used to determine the K_L–K_S mass difference by a method where neutral-kaon strangeness oscillations are monitored through kaon strong interactions, rather than semileptonic decays, thus requiring no assumptions on CPT invariance for the decay amplitudes. The result, Δm=(0.5343±0.0063_stat±0.0025_syst)×10¹⁰ℏ/s, provides a valuable input for CPT tests.

Minimal absent words (MAW) of a genomic sequence are subsequences that are absent themselves but the subwords of which are all present in the sequence. The characteristic distribution of genomic MAWs as a function of their length has been observed to be qualitatively similar for all living organisms, the bulk being rather short, and only relatively few being long. It has been an open issue whether the reason behind this phenomenon is statistical or reflects a biological mechanism, and what biological information is contained in absent words. % In this work we demonstrate that the bulk can be described by a probabilistic model of sampling words from random sequences, while the tail of long MAWs is of biological origin. We introduce the novel concept of a core of a minimal absent word, which are sequences present in the genome and closest to a given MAW. We show that in bacteria and yeast the cores of the longest MAWs, which exist in two or more copies, are located in highly conserved regions the most prominent example being ribosomal RNAs (rRNAs). We also show that while the distribution of the cores of long MAWs is roughly uniform over these genomes on a coarse-grained level, on a more detailed level it is strongly enhanced in 3' untranslated regions (UTRs) and, to a lesser extent, also in 5' UTRs. This indicates that MAWs and associated MAW cores correspond to fine-tuned evolutionary relationships, and suggest that they can be more widely used as markers for genomic complexity.

We describe a new way to calculate the electrostatic properties of macromolecules which eliminates the assumption of a constant dielectric value in the solvent region, resulting in a Generalized Poisson-Boltzmann-Langevin equation (GPBLE). We have implemented a web server (http://lorentz.immstr.pasteur.fr/pdb_hydro.php) that both numerically solves this equation and uses the resulting water density profiles to place water molecules at preferred sites of hydration. Surface atoms with high or low hydration preference can be easily displayed using a simple PyMol script, allowing for the tentative prediction of the dimerization interface in homodimeric proteins, or lipid binding regions in membrane proteins. The web site includes options that permit mutations in the sequence as well as reconstruction of missing side chain and/or main chain atoms. These tools are accessible independently from the electrostatics calculation, and can be used for other modeling purposes. We expect this web server to be useful to structural biologists, as the knowledge of solvent density should prove useful to get better fits at low resolution for X-ray diffraction data and to computational biologists, for whom these profiles could improve the calculation of interaction energies in water between ligands and receptors in docking simulations.

An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron micros-copy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.

Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH). However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs). The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms) and computationally efficient (i.e. with a minimal set of states and transitions). The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9). By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane potential). The kinetic model, developed in the NEURON simulation environment, appears to be the simplest and most parsimonious way for a detailed phenomenological description of the human VGSCs electrophysiological behaviour.

Air-filled polyvinyl alcohol microbubbles (PVA-MBs) were recently introduced as a contrast agent for ultrasound imaging. In the present study, we explore the possibility of extending their application in multimodal imaging by labeling them with a near infrared (NIR) fluorophore, VivoTag-680. PVA-MBs were injected intravenously into FVB/N female mice and their dynamic biodistribution over 24 h was determined by 3D-fluorescence imaging co-registered with 3D-mu CT imaging, to verify the anatomic location. To further confirm the biodistribution results from in vivo imaging, organs were removed and examined histologically using bright field and fluorescence microscopy. Fluorescence imaging detected PVA-MB accumulation in the lungs within the first 30 min post-injection. Redistribution to a low extent was observed in liver and kidneys at 4 h, and to a high extent mainly in the liver and spleen at 24 h. Histology confirmed PVA-MB localization in lung capillaries and macrophages. In the liver, they were associated with Kupffer cells; in the spleen, they were located mostly within the marginal-zone. Occasional MBs were observed in the kidney glomeruli and interstitium. The potential application of PVA-MBs as a contrast agent was also studied using ultrasound (US) imaging in subcutaneous and orthotopic pancreatic cancer mouse models, to visualize blood flow within the tumor mass. In conclusion, this study showed that PVA-MBs are useful as a contrast agent for multimodal imaging. (C) 2015 Elsevier Inc. All rights reserved.

Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 +/- 12 nm diameter, and 40 nm domains were estimated to 65 +/- 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.

Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further.

Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca²⁺ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016.

Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na⁺, K⁺ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems.

In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact.

Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the ability to quantitatively monitor endogenous and exogenous protein expression competition on the single molecule level. Through incorporation of an N-terminal hemagglutinin (HA) epitope to amMaple3 fused Na,K-ATPase (α1 isoform), using PALM and STORM imaging we investigatethe increase in plasma membrane density at the cost of competitive expression. Quantification of plasma membrane protein density revealed a time dependent increase over time of totalprotein content. Results show that plasma membrane densities increased by more than 60%,comparing 17h and 41h transfection times, whilst endogenous levels were simultaneously reduced by 20 %.

The role of the Bcl-family proteins in the mitochondrial apoptotic process is well described with biochemical and molecular methods in studies of isolated mitochondria and transfected cell lines. There is however little knowledge about the mechanisms for Bcl protein interaction leading to apoptosis in intact cells. In particular, the time sequence and location for Bcl protein interaction has so far only been described in hypothetical models.Here we have used Stimulated Emission Depletion (STED) microscopy and Single Molecule Localization Microscopy (SMLM) to study the apoptotic process in immune-stained rat renal epithelia cells exposed to 20 mM glucose (HG) and to study its rescue by ouabain. To assess distance between Bcl-2 proteins, we used the nearest-neighbor algorithm. The anti-apoptotic protein Bcl-xLl was predominantly expressed on mitochondria in control cells, and remained so throughout the process, although its abundance decreased. After 2h HG the apoptosis-inducing protein BAD had translocated from the cytoplasm to the mitochondria where it clustered with Bcl-xL. This occurred before an increase in reactive oxygen species and was dependent on activation of the PI3K –AKT pathway. According to current concepts, Bcl-xL interacts with the apoptotic protein Bax on the mitochondria under control conditions to translocate Bax back to the cytosol1. We found that Bax started to accumulate on the mitochondria after 4h HG and, surprisingly, that the interaction between Bcl-xL and Bax became more pronounced during the course of the apoptotic process. After 6h HG Bax also interacted with the non-specific ion transporter VDAC; an interaction described to lead to penetration of the inner mitochondrial membrane and mark the point of no return.

Background: Anesthetics mediate portions of their activity via modulation of the ?-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. Methods: The structure of the torpedo acetylcholine receptor (nAChR?), the structures of the ?4 and ?2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. Results: Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between ? and ? subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. Conclusion: Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.

Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

Introduction: We have previously used molecular modeling techniques combined with experimental data to visualize a plausible model of an anesthetic binding site within a LGIC complex.¹ We have also previously shown a computational mechanism by which these ion channels may open and close and postulated how this motion may be affected by the presence of anesthetics.² The difficulties with these methods, however, lay in their inability to account for the modest effects of a separate anesthetic ligand or small mutation on ion channel motion. Here we show the successful application of an elastic network calculation on a homologue of the extracellular component of LGIC's, the acetycholine binding protein (AChBP), in the presence and absence of large cobratoxin ligands. These calculations demonstrate a clear alteration in the native symmetric motion of a protein due to the presence of multiple ligands, as may occur with anesthetics and muscle relaxants.

Methods: Coordinates of the AChBP with (1YI5)³ and without (1I9B)⁴ cobratoxin were obtained from the Research Collaboratory for Structural Biology (RCSB). Hydrogens were added using DSViewer 5.0 (Accelrys, San Diego, CA). Normal mode analysis was performed using an all atom elastic network model developed by Lindahl. Root-mean-square deviations (RMSD) of each residue were produced from the application of the RMSD analysis utility within the GROMACS software suite to the coordinate trajectory output files. The RMSD data was then imported into Microsoft Excel for plotting and further comparison of protein backbone motions between the two different normal mode trajectories.

Results: Normal mode analysis reveals that ligand binding to this protein alters its natural harmonic vibration. In this case, the axially symmetric motion of the AChBP, that may be associated with channel gating in the full nAChR, is highly dampened by the presence of bound cobratoxin. A large proportion of the kinetic energy within this mode seems to be absorbed by the cobratoxin, leaving the channel motion significantly decreased.

Conclusions: This is among the first descriptions of the effect of bound ligand on large scale protein dynamics, especially as it relates to ion channel gating. This analysis was possible using an elastic network approximation due to the large protein nature of the cobratoxin ligand. For nonpeptide drugs such as anesthetics which contain far fewer atoms, using the effects of bound ligand on protein motion as additional criteria for future drug design may require a more robust molecular mechanics treatment of the ligand-receptor complex.

Predicting collective dynamics and structural changes in biological macromolecules is pivotal toward a better understanding of many biological processes. Limitations due to large system sizes and inaccessible time scales have prompted the development of alternative techniques for the calculation of such motions. In this work, we present the results of a normal-mode analysis technique based on molecular mechanics that enables the calculation of accurate force-field based vibrations of extremely large molecules and compare it with two elastic network approximate models. When applied to the glycine alpha1 receptor, all three normal-mode analysis algorithms demonstrate an "iris-like" gating motion. Such gating motions have implications for understanding the effects of anesthetic and other ligand binding sites and for the means of transducing agonist binding into ion channel opening. Unlike the more approximate methods, molecular mechanics based analyses can also reveal approximate vibrational frequencies. Such analyses may someday allow the use of protein dynamics elucidated via normal-mode calculations as additional endpoints for future drug design.

Introduction: We have previously used molecular modeling combined with experimental data to visualize a plausible model of an anesthetic binding site within a LGIC.¹ We have also previously shown a computational mechanism by which these LGICs may gate and postulated how this motion may be affected by the presence of anesthetics.² The initial difficulty with these calculations concerns the 26000 atoms present in the receptor and the computing capabilities required to perform vibrational analyses on such a large construct. Here we show the successful application of an elastic network calculation on our previously published model of a glycine alpha one receptor (GlyRa1), now suspended in a fully hydrated lipid bilayer. Despite the presence of over 100,000 atoms , these calculations continue to demonstrate a symmetric motion of the ion channel protein that is consistent with the gating motion demonstrated in previous in vacuo work by us and others. Methods: Coordinates of the GlyRa1 model were obtained from our previous work. A 100x100A lipid bilayer matrix was constructed from POPC and then hydrated on both surfaces with water molecules using the VMD 1.86 software package (NCSA, Urbana, Ill.). Discovery Studio 1.7 (Accelrys, San Diego, CA) molecular modeling software was used to insert our GlyRa1 model into the lipid bilayer such that the known interfacial residue GLY 221 was at the POPC-water interface. All waters within 3.8A of the protein were removed as were all lipid molecules within 2A of the protein. Hydrogens were added followed by energy minimization of the entire system to remove energetically unfavorable contacts. The system was subsequently further hydrated within the GROMACS software suite and subjected to further energy equilibration via molecular dynamics simulation with periodic boundary conditions. Subsequent normal mode analysis was performed using an all atom elastic network model developed by Lindahl which takes advantage of a sparse matrix implementation for computational efficiency. Results: Despite the large size of the system, the introduction of water and lipid did not grossly distort the overall gating motion of the glyRa1 noted in previous works. Normal mode analysis revealed that the GlyRa1 in a fully hydrated bilayer environment continues to demonstrate an iris-like gating motion as a low frequency, high amplitude natural harmonic vibration. Furthermore, the introduction of periodic boundary conditions allowed simultaneous harmonic vibrations of lipid in sync with the protein gating motion that are compatible with reasonable lipid bilayer perturbations. Conclusions: This is among the first description of a normal mode calculation describing large-scale protein dynamics and ion channel gating in the presence of a fully hydrated lipid bilayer complex. This analysis was only possible on such a large system due to the computational efficiencies of the elastic network approximation. This model will hopefully provide a more accurate means of introducing anesthetics and alcohols into protein and lipid bilayer systems and allow us to discern their effects on LGIC gating. ¹Bertaccini EJ, Shapiro J, Brutlag DL, Trudell JR: J Chem Inf Model 2005; 45: 128-35; ²Bertaccini EJ, Trudell JR, Lindahl E:J Chem Inf Model 2007; 47: 1572-9.

Introduction : Ligand-gated ion channels (LGICs) are thought to mediate a significant proportion of anesthetic effects. We built atomic level models of the glycine alpha one receptor (GlyRa1) to examine its interactions with anesthetics. We previously built models of a GlyRa1 based on a prokaryotic pentameric ion channel in the closed state from Erwinia Chrysanthemi (ELIC) (1-3). Here, we built a GlyRa1 model based on the open state structures of two new ion channels from the prokaryote Gloebacter violaceus (GLIC).(4-5) These new templates are relevant since anesthetics are thought to bind to and stabilize the open state of the GlyRa1. Methods : The 3D coordinates of two forms of GLIC (3EHZ.pdb and 3EAM.pdb) were obtained from the RCSB database. The sequence of the human GlyRa1 was obtained from the NCBI database. A BLAST sequence search was performed using the GLIC sequences. Among the best scored homologous human sequences were those of the GlyRa1. The template structures and the sequence of GlyRa1 were aligned with Discovery Studio 2.0.1 (Accelrys, San Diego, CA) and the Modeler module was used for assignment of coordinates for aligned amino acids, the construction of possible loops, and the initial refinement of amino acid sidechains. Results : The BLAST derived scores suggest a close homology between the LGICs, GLIC and ELIC. Subsequent CLUSTALW alignment of the GLIC and GlyRa1 sequences demonstrates reasonable sequence similarity. The model of the GlyRa1 is a homomer with pentameric symmetry about a central ion pore and shows significant transmembrane alpha helical and extracellular beta sheet content. Unlike our previous model based on the ELIC template, the current model based on the GLIC templates shows a continuously open pore with a partial restriction within the transmembrane region. Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3) (ILE229, SER 267, ALA 288). They now line the intersubunit interface, in contrast to our previous models. However, residues from TM4 that are known to modulate a variety of anesthetic effects on this or homologous LGICs are present but could only indirectly influence an intersubunit anesthetic binding site. Normal mode analyses show an iris-like motion similar to previous results.Conclusions : A model of the GlyRa1 was constructed using homology modeling based on the GLIC templates. This model posits an intersubunit site for anesthetic binding that may communicate with the intrasubunit region of each TMD. 

Ligand-gated ion channels (LGICs) significantly modulate anesthetic effects. Their exact molecular structure remains unknown. This has led to ambiguity regarding the proper amino acid alignment within their 3D structure and, in turn, the location of any anesthetic binding sites. Current controversies suggest that such a site could be located in either an intra- or intersubunit locale within the transmembrane domain of the protein. Here, we built a model of the glycine alpha one receptor (GlyRa1) based on the open-state structures of two new high-resolution ion channel templates from the prokaryote, Gloebacter violaceus (GLIC). Sequence scoring suggests reasonable homology between GlyRa1 and GLIC. Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3): (ILE229, SER 267, and ALA 288). They line an intersubunit interface, in contrast to previous models. However, residues from the fourth transmembrane domain (TM4) that are known to modulate a variety of anesthetic effects are quite distant from this putative anesthetic binding site. While this model can account for a large proportion of the physicochemical data regarding such proteins, it cannot readily account for the alterations on anesthetic effects that are due to mutations within TM4.

Introduction: Analyses of ligand-gated ion channel receptors (LGIC) have demonstrated that possible sites of anesthetic action exist within their transmembrane domains. We have previously used molecular modeling techniques combined with experimental data to visualize a plausible model of an anesthetic binding site within a LGIC complex.¹ We have also previously shown an approximate computational mechanism, based on an elastic network model, by which these ion channels may open and close and postulated how this motion may be affected by the presence of anesthetics.² The difficulties with these approximation methods, however, lay in their inability to account for the modest effects of a separate ligand or a small mutation on ion channel motion. Here we show the successful application of a formal molecular mechanics force field for the normal mode calculation of protein motions.Methods: Coordinates of the homomeric GABARa1 pentamer complex composed of both an extracellular ligand binding domain and a transmembrane domain came from our previous work.³ Using this structure as a template, we built a model of the glyRa1 homomer using the homology modeling tools within the InsightII 2005 software package (Accelrys, San Diego, CA). This model then underwent a series of restrained optimizations within the GROMACS modeling package using the OPLS force field and no distance cutoffs on electrostatic and van der Waals interactions. After final unrestrained optimization, normal mode analysis was performed with a sparse matrix implementation.Results: As we previously reported for the approximate elastic network technique², analysis of the entire glyRa1 complex demonstrated a clear iris-like motion of the protein about the central axis of the ion pore as the first (highest amplitude-lowest frequency) normal mode. In this mode, the rotation of the ligand binding domain occurred in the opposite direction to that of the transmembrane domain, producing a “wringing” like motion of the entire protein complex as it traversed its gating cycle. However, unlike the elastic network calculation of normal modes, which could only report relative frequencies of vibration, the GROMACS-based normal mode analysis allows for the calculation of real vibrational frequencies on the order of 321 GHz or around 3.1 ps per cycle. Likewise, while elastic network calculations could be completed in a few hours, the GROMACS calculations took approximately a week to complete on a Dell Workstation with dual 3GHz Xeon processors and a 64 bit software implementation.Conclusions: Despite these proteins containing upwards of 26,000 atoms, our new methods have made it possible to derive normal modes via the full implementation of a formal force field calculation. Despite their length and markedly increased complexity, these calculations still demonstrate that the harmonic motion of LGIC complexes is consistent with the direction of channel opening and closing. Such calculations should now allow the elucidation of the subtle effects on ion channel motion that are due to anesthetic binding.

CHARMM27 is a widespread and popular force field for biomolecular simulation, and several recent algorithms such as implicit solvent models have been developed specifically for it. We have here implemented the CHARMM force field and all necessary extended functional forms in the GROMACS molecular simulation package, to make CHARMM-specific features available and to test them in combination with techniques for extended time steps, to make all major force fields available for comparison studies in GROMACS, and to test various solvent model optimizations, in particular the effect of Lennard-Jones interactions on hydrogens. The implementation has full support both for CHARMM-specific features such as multiple potentials over the same dihedral angle and the grid-based energy correction map on the phi, psi protein backbone dihedrals, as well as all GROMACS features such as virtual hydrogen interaction sites that enable 5 fs time steps. The medium-to-long time effects of both the correction maps and virtual sites have been tested by performing a series of 100 ns simulations using different models for water representation, including comparisons between CHARMM and traditional TIP3P. Including the correction maps improves sampling of near native-state conformations in our systems, and to some extent it is even able to refine distorted protein conformations. Finally, we show that this accuracy is largely maintained with a new implicit solvent implementation that works with virtual interaction sites, which enables performance in excess of 250 ns/day for a 900-atom protein on a quad-core desktop computer.

Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins-not to mention numerous applications in drug design. Here, we present a full 1 micros atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120 degrees rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation ( approximately 35 degrees ) of the extracellular end of all S4 segments is present also in a reference 0.5 micros simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 3(10) helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4-lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5-1 micros). Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations.

High resolution Stimulated Emission Depletion (STED) microscopy has been demonstrated for fundamental studies in cells, living tissue and organisms. Today, a major trend in the STED technique development is to make the instruments simpler and more user-friendly, without compromising performance. This has become possible by new low-cost, turn-key laser technology and by implementing specifically designed phase plates and polarization elements, extending and simplifying the shaping of the laser beam profiles. These simpler and cheaper realizations of STED are now becoming more broadly available. In parallel with the continuous development of sample preparation and fluorophore reporter molecules ultimately setting the limit of the image quality, contrast and resolution, we can thus expect a significant increase in the use of STED, in science as well as for clinical and drug development purposes.

Despite its short history, diffraction-unlimited fluorescence microscopy techniques have already made a substantial imprint in the biological sciences. In this review, we describe how stimulated emission depletion (STED) imaging originally evolved, how it compares to other optical super-resolution imaging techniques, and what advantages it provides compared to previous golden-standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. We outline the prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics, and describe major evolving strategies for how to push the borders of STED imaging even further in life science. Finally, we provide examples of how STED nanoscopy can be applied, within three different fields with particular potential for STED imaging experiments: neuroscience, plasma membrane biophysics, and subcellular clinical diagnostics. In these areas, and in many more, STED imaging can be expected to play an increasingly important role in the future.

Lipid molecules form thin biological membranes that envelop all living cells, and behave as two-dimensional liquid sheets immersed in bulk water. The interactions of such biomembranes with their environment lay the foundation of a plethora of biological processes rooted in the mesoscopic domain - length scales of 1-1000 nm and time scales of 1-1000 ns. Research in this intermediate regime has for a long time been out of reach for conventional experiments, but breakthroughs in computer simulation methods and scattering experimental techniques have made it possible to directly probe static and dynamic properties of biomembranes on these scales.

Biomembranes are soft, with a relatively low energy cost of bending, and are thereby influenced by random, thermal fluctuations of individual molecules. Molecular dynamics simulations show how in-plane (density fluctuations) and out-of-plane (undulations) motions are intertwined in the bilayer in the mesoscopic domain. By novel methods, the fluctuation spectra of lipid bilayers can be calculated withdirect Fourier analysis. The interpretation of the fluctuation spectra reveals a picture where density fluctuations and undulations are most pronounced on different length scales, but coalesce in the mesoscopic regime. This analysis has significant consequences for comparison of simulation data to experiments. These new methods merge the molecular fluctuations on small wavelengths, with continuum fluctuations of the elastic membrane sheet on large wavelengths, allowing electron density profiles (EDP) and area per lipid to be extracted from simulations with high accuracy.

Molecular dynamics simulations also provide insight on the small-wavelength dynamics of lipid membranes. Rapidly decaying density fluctuations can be described as propagating sound waves in the framework of linearized hydrodynamics, but there is a slow, dispersive, contribution that needs to be described by a stretched exponential over a broad range of length- and time scales - recent experiments suggest that this behavior can prevail even on micrometer length scales. The origin of this behavior is discussed in the context of fluctuations of the bilayer interface and the molecular structure of the bilayer itself. Connections to recent neutron scattering experiments are highlighted.

Atomic resolution and coarse-grained simulations of dimyristoylphosphatidylcholine lipid bilayers were analyzed for fluctuations perpendicular to the bilayer using a completely Fourier-based method. We find that the fluctuation spectrum of motions perpendicular to the bilayer can be decomposed into just two parts: 1), a pure undulation spectrum proportional to q(-4) that dominates in the small-q regime; and 2), a molecular density structure factor contribution that dominates in the large-q regime. There is no need for a term proportional to q(-2) that has been postulated for protrusion fluctuations and that appeared to have been necessary to fit the spectrum for intermediate q. We suggest that earlier reports of such a term were due to the artifact of binning and smoothing in real space before obtaining the Fourier spectrum. The observability of an intermediate protrusion regime from the fluctuation spectrum is discussed based on measured and calculated material constants.

The traditional method for extracting electron density and other transmembrane profiles from molecular dynamics simulations of lipid bilayers fails for large bilayer systems, because it assumes a flat reference surface that does not take into account long wavelength undulations. We have developed what we believe to be a novel set of methods to characterize these undulations and extract the underlying profiles in the large systems. Our approach first obtains an undulation reference surface for each frame in the simulation and subsequently isolates the long-wavelength undulations by filtering out the intrinsic short wavelength modes. We then describe two methods to obtain the appropriate profiles from the undulating reference surface. Most combinations of methods give similar results for the electron density profiles of our simulations of 1024 DMPC lipids. From simulations of smaller systems, we also characterize the finite size effect related to the boundary conditions of the simulation box. In addition, we have developed a set of methods that use the undulation reference surface to determine the true area per lipid which, due to undulations, is larger than the projected area commonly reported from simulations.

The classical view of neuronal protein synthesis is that proteins are made in the cell body and then transported to their functional sites in the dendrites and the dendritic spines. Indirect evidence, however, suggests that protein synthesis can directly occur in the distal dendrites, far from the cell body. We are developing protocols for dual labeling of RNA and proteins using (15)N-uridine and (18)O- or (13)C-leucine pulse chase in cultured neurons to identify and localize both protein synthesis and fate of newly synthesized proteins. Pilot experiments show discrete localization of both RNA and newly synthesized proteins in dendrites, close to dendritic spines. We have for the first time directly imaged and measured the production of proteins at the subcellular level in the neuronal dendrites, close to the functional sites, the dendritic spines. This will open a powerful way to study neural growth and synapse plasticity in health and disease.

The estimation of body fluids is a useful and common practice for assessment of disease status and therapy outcomes. Electrical bioimpedance spectroscopy (EBIS) methods are noninvasive, inexpensive and efficient alternatives for determination of body fluids. One of the main source of errors in EBIS measurements in the estimation of body fluids is capacitive coupling. In this paper an analysis of capacitive coupling in EBIS measurements was performed and the robustness of the different immittance spectra against it tested. On simulations the conductance (G) spectrum presented the smallest overall error, among all immittance spectra, in the estimation of the impedance parameters used to estimate body fluids. Afterwards the frequency range of 10-500 kHz showed to be the most robust band of the G spectrum. The accuracy of body fluid estimations from the resulting parameters that utilized G spectrum and parameters provided by the measuring device were tested on EBIS clinical measurements from growth hormone replacement therapy patients against estimations performed with dilution methods. Regarding extracellular fluid, the correlation between each EBIS method and dilution was 0.93 with limits of agreement of 1.06 +/- 2.95 l for the device, 1.10 +/- 2.94 l for G [10-500 kHz] and 1.04 +/- 2.94 l for G [5-1000 kHz]. Regarding intracellular fluid, the correlation between dilution and the device was 0.91, same as for G [10-500 kHz] and 0.92 for G [5- 1000 kHz]. Limits of agreement were 0.12 +/- 4.46 l for the device, 0.09 +/- 4.45 for G [10- 500 kHz] and 0.04 +/- 4.58 for G [5-1000 kHz]. Such close results between the EBIS methods validate the proposed approach of using G spectrum for initial Cole characterization and posterior clinical estimation of body fluids status.

A biophysical model of a neocortical microcircuit system is formulated and employed in studies of neuromodulatory control of dynamics and function. The model is based on recent observations of reciprocal connections between pyramidal cells and inhibitory interneurons and incorporates a new type of activity-dependent short-term depression of synaptic couplings recently observed. The model neurons are of a low-dimensional type also accounting for neuronal adaptation, i.e. the coupling between neuronal activity and excitability, which can be regulated by various neuromodulators in the brain. The results obtained demonstrate a capacity for neuromodulatory control of dynamical mode linked to functional mode. The functional aspects considered refer to the observed resolution of multiple objects in working memory as well as the binding of different features for the perception of an object. The effects of neuromodulators displayed by the model are in accordance with many observations on neuromodulatory influence on cognitive functions and brain disorders.

The κ opioid receptor (κOR) is a member of G-protein-coupled receptors, and is considered as a promising drug target for treating neurological diseases. κOR selective 6′-GNTI was proved to be a G-protein biased agonist, whereas 5′-GNTI acts as an antagonist. To investigate the molecular mechanism of how these two ligands induce different behaviors of the receptor, we built two systems containing the 5′-GNTI-κOR complex and the 6′-GNTI-κOR complex, respectively, and performed molecular dynamics simulations of the two systems. We observe that transmembrane (TM) helix 6 of the κOR rotates about 4.6° on average in the κOR-6′-GNTI complex. Detailed analyses of the simulation results indicate that E2976.58 and I2946.55 play crucial roles in the rotation of TM6. In the simulation of the κOR-5′-GNTI system, it is revealed that 5′-GNTI can stabilize TM6 in the inactive state form. In addition, the kink of TM7 is stabilized by a hydrogen bond between S3247.47 and the residue V691.42 on TM1.

In this work, fluorescence correlation spectroscopy (FCS) was used to investigate the effects of potassium iodide (KI) on the electronic-state population kinetics of a range of organic dyes in the visible wavelength range. Apart from a heavy atom effect promoting intersystem crossing to the triplet states in all dyes, KI was also found to enhance the triplet-state decay rate by a charge-coupled deactivation. This deactivation was only found for dyes with excitation maximum in the blue range, not for those with excitation maxima at wavelengths in the green range or longer. Consequently, under excitation conditions sufficient for triplet state formation, KI can promote the triplet state buildup of one dye and reduce it for another, red-shifted dye. This anticorrelated, spectrally separable response of two different dyes to the presence of one and the same agent may provide a useful readout for biomolecular interaction and microenvironmental monitoring studies. In contrast to the typical notion of KI as a fluorescence quencher, the FCS measurements also revealed that when added in micromolar concentrations KI can act as an antioxidant, promoting the recovery of photo-oxidized fluorophores. However, in millimolar concentrations KI also reduces intact, fluorescently viable fluorophores to a considerable extent. In aqueous solutions, for the dye Rhodamine Green, an optimal concentration of KI of approximately 5 mM can be defined at which the fluorescence signal is maximized. This concentration is not high enough to allow full triplet state quenching. Therefore, as a fluorescence enhancement agent, it is primarily the antioxidative properties of KI that play a role.

For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the buildup of photoinduced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules. The concept was experimentally verified by measuring the fluorescence response of freely diffusing cyanine fluorophores (Cy5), undergoingtrans-cis isomerization when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.

Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field.

By a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST)imaging, the triplet state kinetics of the membrane fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl(NBD) was studied in small unilamellar vesicles (SUVs). It was shown that spin-labels, included inSUV membranes together with the NBD-labeled lipids, induce prominent effects on the triplet statekinetics of NBD. The relative effects on the triplet state kinetics are considerably larger than thoseobserved in traditional fluorescence quenching studies, and can provide information about thelocalization and the interactions between the lipids in the SUVs, using considerably lowerconcentrations of spin-labeled lipids in the membranes. From the effects of the spin labels on thetriplet state kinetics of NBD, we revisited the folding behavior of NBD-labeled phospholipid chains inthe membranes. Our results indicate that the NBD probe on the acyl chain of the phospholipids do notunambiguously loop back towards the membrane surface, as previously reported, but may alternatebetween a straight and a folded acyl chain, with the NBD label at the surface, or deep into themembrane bilayer. Our study suggests that the triplet state parameters of NBD can provide anadditional set of orthogonal parameters, which can increase accuracy and precision of fluorescencebasedmolecular dynamics and interaction studies with NDB as a probe molecule.

We demonstrate a method for investigating natural killer (NK) cell aggression against ultrasound-assisted human hepatocellular carcinoma (HCC) HepG2 solid tumors in a multi-well microdevice. We quantify the activity of human primary IL-2 activated NK cells against HepG2 tumors for up to five days and we present the correlation between NK cell numbers versus average tumor volume and final tumor outcome (growth or defeat). We suggest future applications on formation of tumors originated from primary tumors cells and other tumor components as well as primary NK originating from the patient for use in personalized immunotherapy.

Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.

Whilst rigid, planar surfaces are often used to study cell migration, a physiological scenario requires three-dimensional (3D) scaffolds with tissue-like stiffness. This paper presents a method for fabricating periodic hydrogel scaffolds with a 3D honeycomb-like structure from colloidal crystal templates. The scaffolds, made of hydrogel-walled cavities interconnected by pores, have separately tuneable internal dimensions and adjustable gel stiffness down to that of soft tissues. In conjunction with confocal microscopy, these scaffolds were used to study the importance of cell compliance on invasive potential. Acute promyelocytic leukaemia (APL) cells were differentiated with all-trans retinoic acid (ATRA) and treated with paclitaxel. Their migration ability into the scaffolds' size-restricted pores, enabled by cell softening during ATRA differentiation, was significantly reduced by paclitaxel treatment, which interferes with cell shape recovery. These findings demonstrate the usability of the scaffolds for investigating factors that affect cell migration, and potentially other cell functions, in a realistic 3D tissue model.

Compressible large eddy simulation is employed to numerically investigate the laryngeal flow. Symmetric static models of the human larynx with a divergent glottis are considered, with the presence of false vocal folds (FVFs). The compressible study agrees well with that of the incompressible study. Due to the high enough Reynolds number, the flow is unsteady and develops asymmetric states downstream of the glottis. The glottal jet curvature decreases with the presence of FVFs or the ventricular folds. The gap between the FVFs stretches the flow structure and reduces the jet curvature. The presence of FVFs has a significant effect on the laryngeal flow resistance. The intra-glottal vortex structures are formed on the divergent wall of the glottis, immediately downstream of the separation point. The vortices are then convected downstream and characterized by a significant negative static pressure. The FVFs are a main factor in the generation of stronger vortices, and thus on the closure of the TVFs. The direct link between the FVFs geometry and the motion of the TVFs, and by extension to the voice production, is of interest for medical applications as well as future research works. The presence of the FVFs also changes the dominant frequencies in the velocity and pressure spectra.

Motion analysis is a common clinical assessment and research tool that uses a camera system or motion sensors and force plates to collect kinematic and kinetic information of a subject performing an activity of interest. The use of force plates can be challenging and sometimes even impossible. Over the past decade, several computational methods have been developed that aim to preclude the use of force plates. Useful in particular for predictive simulations, where a new motion or change in control strategy inherently means different external contact loads. These methods, however, often depend on prior knowledge of common observed ground reaction force (GRF) patterns, are computationally expensive, or difficult to implement. In this study, we evaluated the use of the Zero Moment Point as a computationally inexpensive tool to obtain the GRFs for normal human gait. The method was applied on ten healthy subjects walking in a motion analysis laboratory and predicted GRFs are evaluated against the simultaneously measured force plate data. Apart from the antero-posterior forces, GRFs are well-predicted and errors fall within the error ranges from other published methods. Joint extension moments were underestimated at the ankle and hip but overestimated at the knee, attributable to the observed discrepancy in the predicted application points of the GRFs. The computationally inexpensive method evaluated in this study can reasonably well predict the GRFs for normal human gait without using prior knowledge of common gait kinetics.