Paper: Modeling Subunit Cooperativity in Opening of Tetrameric Ion Channels
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Helical content of Protein DataBank entries
From the Marshall lab: Helices are the most abundant secondary structural element in proteins, and are integral to a variety of protein-based phenomena. We analyzed the hydrogen bond content of the Protein DataBank to show that the population of helices is somewhat different than the population predicted by classic ideal forms of helix, such as alpha- and 3(10)- forms. This observation, which is strictly derived from high-resolution atomic coordinates, enables a detailed view of structural features in helices. We apply these constraints to the design and synthesis of novel peptides and helical peptidomimetics.
Determination of the interface for hTRF2 with telomeric DNA using H/D amid exchange mass spectrometry methods
From the M. Gross Lab: One goal is to develop and apply mass-spectrometry-based methods for foot printing proteins, taking advantage of mass-spectrometry based proteomics analysis. An example is H/D amide exchange to follow conformational changes in proteins, determine their interfaces in binding with other proteins, and quantify the binding affinities of proteins with ligands (PLIMSTEX). Exchange followed by protein digestion, usually with pepsin at low pH (quench conditions) reveals those regions that change upon binding. Examples are the determination of the interface for hTRF2 (human telomeric repeat binding factor) with telomeric DNA (Biochemistry, 47, 1797-1807 (2008)--see graphic) and the identification of the interface of a toxic protein, SPN, from the bacterial pathogen Streptococcus pyogenes with IFS. A complementary foot printing approach is fast photochemical oxidation of proteins (FPOP) whereby OH radicals are laser-generated rapidly in a flow system and react on the microsec time scale with solvent accessible aromatic and sulfur-containing amino-acid residues, thus mapping the protein surface.
Cancer treatment with avb3-targeted fumagillin nanoparticles
From the paper: Treatment with avb3-targeted fumagillin nanoparticles diminished the development of neovasculature and reduced early Vx-2 tumor growth in rabbits with drug levels orders of magnitude lower than those previously used in rodent experiments and human clinical trials. Quantitative MR molecular imaging with anb3-targeted paramagnetic nanoparticles revealed that the neovasculature was predominately located in the peripheral aspects of the tumor, which was corroborated by the co-localization of avb3-targeted rhodamine nanoparticles with FITC-lectin exclusively in the tumor periphery. High-resolution three-dimensional neovascular maps further illustrated the asymmetric, patchy pattern of angiogenic expression, which was diminished by anb3-targeted fumagillin nanoparticles. anb3-targeted fumagillin nanoparticles may provide a safer and more effective approach to deliver potent MetAP2 inhibitors.
Understanding the role of proline isomerization in ribonuclease T1 folding
From the Frieden lab: Protein folding and aggregation are under study in this laboratory. The long-term goal of the folding work is to understand the nature of the intermediate structures on the unfolding and refolding pathways. Work in the laboratory uses site-directed mutagenesis and techniques such as 19F and proton NMR, circular dichroism, fluorescence measurements and other biophysical methods. Current studies include the incorporation of fluoroproline into proteins to examine the role of proline isomerization in folding as followed by NMR. Shown here is one such protein under study, ribonuclease T1. The laboratory is also studying, by the same techniques, the properties of those proteins and peptides characterized as intrinsically disordered. These peptides/proteins are involved with the formation of aggregates found in diseases such as Alzheimer’s, Parkinson’s and Huntington’s.
Metal binding by RcnR
From the Chivers lab: First-row transition metals are essential for life in all organisms. However, they can be toxic in excess and must be precisely regulated. We focus on structure-function relationships in proteins involved in nickel acquisition and intracellular regulation. We recently identified a new nickel- and cobalt-responsive transcriptional regulator, called RcnR, that is the founding member of a new structural class of transcriptional regulators in bacteria. We have used a combination of biochemical, spectroscopic, and biological experiments to probe the metal-binding site of RcnR. We discovered a Co-S interaction that is unique to known cobalt responsive transcriptional regulators. This interaction is important for cobalt selectivity by RcnR.
The ATP-sensitive potassium channel
From the Nichols lab: The ATP-sensitive potassium channel is a key link between metabolism of the cell and its electrical activity. The structure is modeled on a bacterial homolog, and shows the ATP binding site to which ATP binds, favoring the closed state of the channel. (KATP channels as molecular sensors of cellular metabolism. Nichols, C.G., Nature. 2006, 440:470-6). For more on the biophysics of ion channels, visit the Nichols lab web site http://www.nicholslab.wustl.edu/
Mechanisms of protein aggregation
From the Pappu lab: Research in the Pappu lab focuses on mechanisms of protein aggregation. A combination of polymer theory, novel simulations, and experiments are being used. The image shows the result of a first principles simulation of the self-association two intrinsically disordered polyglutamine molecules. Predictions made from analyzing these types of data are yielding insights into the molecular basis of Huntington's disease, which is associated with mutations that lead to polyglutamine expansions. For more details, please visit the Pappu lab at http://lima.wustl.edu.
Cryo-EM reconstruction of virus
From the Smith lab: This is an cryo-electron microscopy image reconstruction of cucumber necrosis virus. The image in the upper left hand corner is a representation of the virus protein capsid with each of the subunits represented by a different color. The large image is a cutaway of the image reconstruction. We found that the virus is composed of three distinct shells that, together with previously published neutron scattering experiments, are composed of protein (the outer blue shell), RNA (the second mauve shell), and protein (the inner orange shell). Mutations in the inner protein shell affects the ability of the virus to package the RNA genome and results in empty, smaller T=1 particles. Katpally, U., Kakani, K., Reade, R., Rochon, D., Smith, T. J. (2007). Structures of T=1 and T=3 Cucumber necrosis virus particles: evidence of internal scaffolding. J. Mol. Biol. 365: 502-512.
Arp2/3 actin complex structure and dynamics
From the Sept lab: The Arp2/3 complex plays a key role in the process of cell motility. In order for Arp2/3 to branch or nucleate new actin filaments, it must first get activated by proteins of the WASp/Scar family. All WASp/Scar proteins contain a series of domains known as VCA that are responsible for this activation, however the molecular details of how this occurs are not known. The picture at the left shows arp3 (blue), arp2 (pink) and an actin monomer (green) bound with the VCA peptide from N-WASp (orange). This structural model is the result of a combination of molecular dynamics and docking simulations, and it agrees very well with experimental findings.
ssDNA interactions with the RecBCD helicase
From the Lohman and Baker labs: Computational models for potential paths for single-stranded DNA through the RecBCD helicase. Wong CJ, Rice RL, Baker NA, Ju T, Lohman TM. Probing 3′-ssDNA Loop Formation in E. coli RecBCD/RecBC–DNA Complexes Using Non-natural DNA: A Model for “Chi” Recognition Complexes. J Mol Biol, 362 (1), 26-43, 2006.
Electrostatic properties of model lipid bilayers
From the Baker lab: We are interested in the electrostatic properties of model lipid bilayers. This image shows the results of molecular dynamics simulations on DPPC bilayer systems. Asymmetric surface potentials are induced by Na+ binding to DPPC bilayers surrounded by solutions of (1) 145 mM KCl and 5 mM NaCl and (2) 145 mM NaCl and 5 mM KCl. Na+ ions shown in blue, K+ ions shown in green, and Cl- ions shown in orange.
Actin filaments moving by Brownian motion
From the Carlsson and Sept labs: Two actin filaments moving by Brownian motion find each other and develop a lasting relationship. This will eventually lead to bundle formation, which is important for the formation of extracellular protrusions such as filopodia (click for animation).
Diffusion simulations of neuromuscular junctions
From the Baker lab: We are interested in developing multiscale computational models to describe cellular phenomena while incorporating information about important biomolecular components. This picture depicts our initial work on modeling syntapic transmission at neuromuscular junctions with a complete description of the important enzyme acetylcholinesterase.
Membrane pore formation by Bax
From the Schlesinger lab: The proapoptotic protein Bax forms pores in the outer mitochondrial membrane that release cytochrome c. Using giant unilamellar liposomes (10-20 microns) filled with fluorescein we have directly visualized the formation of a single Bax pore by the release of the fluorescent dye. The Bax was the genetically engineered human Bax that was activated by removing helix 9.The Bax pore remains open for more than 1 second.
Actin filament polymerization
From the Carlsson lab: Actin filaments are believed to polymerize by a Brownian-ratchet mechanism whereby new monomers "sneak" into gaps that appear between the growing filament and the obstacle it is pushing, due to thermal fluctuations. These simulations show that the Brownian-ratchet mechanism can function even when the filament is attached to the obstacle (click for animation).
Intracellular nickel trafficking and regulation
From the Chivers lab: First-row transition metals are essential for life in all organisms. However, they can be toxic in excess and must be precisely regulated. We focus on the nickel-dependent transcriptional regulator NikR, which is found in a wide variety of microbes. We are interested in various aspects of NikR function including how nickel activates NikR for DNA-binding and how and when NikR senses nickel inside the cell. We use a wide variety of experimental methods ranging from spectroscopic techniques to molecular genetics to address these questions both in the test tube and inside the cell.
Mechanisms of protein folding
From the Frieden lab: The mechanism of protein folding, the role of chaperones in the folding process, the relation of protein structure to function and measurements of protein dynamics are projects under study in this laboratory. The long-term goal of the protein folding work is to understand the nature of the intermediate structures on the unfolding and refolding pathways. Work in the laboratory uses site-directed mutagenesis and techniques such as 19F and proton NMR, circular dichroism, fluorescence measurements and other biophysical methods. Shown here is one of the proteins under study called PapD. This protein is required for the formation of pili in pathogenic bacteria but functions as a chaperone for the folding of other protein subunits that make up the pilus structure. The protein has two distinct domains and real-time NMR experiments show that that there must be domain-domain interaction for correct folding of the protein.
NikR molecular dynamics
From the Chivers and Baker labs: Collaborative work between the Chivers and Baker groups investigating the molecular dynamics of NikR to better understand the molecular basis of its allosteric activity. This figure shows the principal modes of motion of the NikR molecule (foreground) as obtained by analyzing conformational covariance (background) from a molecular dynamics simulation.
Iron responsive element RNA hairpin dynamics
From the Hall lab: The human Iron Response Element RNA hairpin. The six nucleotides in the loop of this stemloop are phylogenetically conserved, and presumably are recognized by the IRE Binding Protein. We use fluorescence and NMR to study the dynamics of the loop nucleotides.
The TINKER molecular modeling software package
From the Ponder lab: Our lab develops and distributes the TINKER molecular modeling software and its associated Force Field Explorer (FFE) GUI. A screenshot from FFE is shown at left. Recent research has centered on the AMOEBA polarizable atomic multipole force field. Initial application to ion solvation and peptide structure indicate that the AMOEBA potential substantially outperforms current generation models used for biomolecular simulation. AMOEBA parameters for nucleic acid systems are under active development. Additional applications involving accurate prediction of protein-ligand binding affinities and generation of high-resolution protein homology models are underway.
Nanoemulsion drug delivery mechanisms
From the Schlesinger lab: Amphipath-stabilized nanoemulsions can be used to deliver drugs and imagining probes to the cell membranes by contact transfer. We have studied the nature of this delivery using electron microscopy. The nano-emulsion can form a hemifusion complex with bilayer membranes. The upper panel shows the hemifusion complex between the smooth bilayer and the nano-emulsion after freeze fracture to reveal the inner leaflet of the liposome. The lower panels are deep etch platinum replicas of nano-emulsions on call membranes. Targeting of the nano-emulsion to integrin receptors increases the nano-emulsion interaction with the microvilli.
Bicarbonate ABC transporter CmpA
From the Smith lab: Shown in the foreground is the structure of a solute binding domain from a bicarbonate ABC transporter from Synechocystis 6803 called CmpA. The red and white molecule in the central 'mouth' of the protein is the bound bicarbonate moiety. In the background are fluorescence microscopy images of Synechocystis. This protein has remarkable selectivity for bicarbonate over nitrate and carbonic acid. Koropatkin, N. M., Koppenaal, D. W., Pakrasi, H. B., Smith, T. J. (2007) The structure of a cyanobacterial bicarbonate transport protein, CmpA. J. Biol. Chem. 282: 2606-2614.
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