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1.  Thermodynamics and Mechanics of Membrane Curvature Generation and Sensing by Proteins and Lipids 
Research investigating lipid membrane curvature generation and sensing is a rapidly developing frontier in membrane physical chemistry and biophysics. The fast recent progress is based on the discovery of a plethora of proteins involved in coupling membrane shape to cellular membrane function, the design of new quantitative experimental techniques to study aspects of membrane curvature, and the development of analytical theories and simulation techniques that allow a mechanistic interpretation of quantitative measurements. The present review first provides an overview of important classes of membrane proteins for which function is coupled to membrane curvature. We then survey several mechanisms that are assumed to underlie membrane curvature sensing and generation. Finally, we discuss relatively simple thermodynamic/mechanical models that allow quantitative interpretation of experimental observations.
PMCID: PMC4205088  PMID: 21219150
curvature-composition coupling; giant unilamellar vesicle; lipid membrane; tube
2.  Membrane Protein Structure and Dynamics from NMR Spectroscopy 
We review the current state of membrane protein structure determination using solid-state nuclear magnetic resonance (NMR) spectroscopy. Multidimensional magic-angle-spinning correlation NMR combined with oriented-sample experiments has made it possible to measure a full panel of structural constraints of membrane proteins directly in lipid bilayers. These constraints include torsion angles, interatomic distances, oligomeric structure, protein dynamics, ligand structure and dynamics, and protein orientation and depth of insertion in the lipid bilayer. Using solid-state NMR, researchers have studied potassium channels, proton channels, Ca2+ pumps, G protein–coupled receptors, bacterial outer membrane proteins, and viral fusion proteins to elucidate their mechanisms of action. Many of these membrane proteins have also been investigated in detergent micelles using solution NMR. Comparison of the solid-state and solution NMR structures provides important insights into the effects of the solubilizing environment on membrane protein structure and dynamics.
PMCID: PMC4082981  PMID: 22136620
magic-angle spinning; multidimensional correlation; solid-state nuclear magnetic resonance; ion channels; GPCR
3.  Biomolecular Imaging with Coherent Nonlinear Vibrational Microscopy 
Optical imaging with spectroscopic vibrational contrast is a label-free solution for visualizing, identifying, and quantifying a wide range of biomolecular compounds in biological materials. Both linear and nonlinear vibrational microscopy techniques derive their imaging contrast from infrared active or Raman allowed molecular transitions, which provide a rich palette for interrogating chemical and structural details of the sample. Yet nonlinear optical methods, which include both second-order sum-frequency generation (SFG) and third-order coherent Raman scattering (CRS) techniques, offer several improved imaging capabilities over their linear precursors. Nonlinear vibrational microscopy features unprecedented vibrational imaging speeds, provides strategies for higher spatial resolution, and gives access to additional molecular parameters. These advances have turned vibrational microscopy into a premier tool for chemically dissecting live cells and tissues. This review discusses the molecular contrast of SFG and CRS microscopy and highlights several of the advanced imaging capabilities that have impacted biological and biomedical research.
PMCID: PMC3965563  PMID: 23245525
vibrational spectroscopy; nonlinear optical microscopy; sum-frequency generation; coherent Raman scattering; biomedical imaging
4.  Dewetting and Hydrophobic Interaction in Physical and Biological Systems 
Hydrophobicity manifests itself differently on large and small length scales. This review focuses on large length scale hydrophobicity, particularly on dewetting at single hydrophobic surfaces and drying in regions bounded on two or more sides by hydrophobic surfaces. We review applicable theories, simulations and experiments pertaining to large scale hydrophobicity in physical and biomoleclar systems and clarify some of the critical issues pertaining to this subject. Given space constraints, we could not review all of the significant and interesting work in this very active field.
PMCID: PMC3898792  PMID: 18928403
Dewetting transition; hydrophobic interaction; protein folding; superhydrophobicity; nano-plates
5.  Functional Motifs in Biochemical Reaction Networks 
The signal-response characteristics of a living cell are determined by complex networks of interacting genes, proteins, and metabolites. Understanding how cells respond to specific challenges, how these responses are contravened in diseased cells, and how to intervene pharmacologically in the decision-making processes of cells requires an accurate theory of the information-processing capabilities of macromolecular regulatory networks. Adopting an engineer’s approach to control systems, we ask whether realistic cellular control networks can be decomposed into simple regulatory motifs that carry out specific functions in a cell. We show that such functional motifs exist and review the experimental evidence that they control cellular responses as expected.
PMCID: PMC3773234  PMID: 20055671
signal transduction; feedback; feed-forward; switches; clocks
6.  Photophysics of Fluorescence Probes for Single Molecule Biophysics and Super-Resolution Imaging 
Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of certain color and brightness but instead have their own ‘personalities’ regarding spectroscopic parameters, redox properties, size and water solubility, photostability and several more. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single molecule biophysical studies which was later overcome using Trolox through reducing/oxidizing system, but a boon for super-resolution imaging due to the controllable photoswitching. Understanding photophysics is critical in design and interpreting single molecule experiments.
PMCID: PMC3736144  PMID: 22404588
FRET; single particle tracking; single molecule stoichiometry; triplet state; redox blinking; photochromic blinking
Combined QM/MM methods provide an accurate and efficient energetic description of complex chemical and biological systems, leading to significant advances in the understanding of chemical reactions in solution and in enzymes. Progress in QM/MM methodology and application will be reviewed, with a focus on ab initio QM based approaches. Ab initio QM/MM methods capitalize on the accuracy and reliability of the associated quantum mechanical approaches, however at a much higher computational cost compared with semiempirical quantum mechanical approaches. Thus reaction path and activation free energy calculations based on ab initio QM/MM methods encounter unique challenges in simulation timescales and phase space sampling. Recent developments overcoming these challenges and enabling accurate free energy determination for reaction processes in solution and enzymes will be featured in this review, along with applications.
PMCID: PMC3727228  PMID: 18393679
enzyme catalysis; solution reaction; enzyme proficiency; conformational dynamics; potential of mean force
8.  Multidimensional Attosecond Resonant X-Ray Spectroscopy of Molecules: Lessons from the Optical Regime 
New free-electron laser and high-harmonic generation X-ray light sources are capable of supplying pulses short and intense enough to perform resonant nonlinear time-resolved experiments in molecules. Valence-electron motions can be triggered impulsively by core excitations and monitored with high temporal and spatial resolution. We discuss possible experiments that employ attosecond X-ray pulses to probe the quantum coherence and correlations of valence electrons and holes, rather than the charge density alone, building on the analogy with existing studies of vibrational motions using femtosecond techniques in the visible regime.
PMCID: PMC3721744  PMID: 23245522
ultrafast; core hole; electron correlation; coherence; stimulated Raman
9.  Superresolution Imaging using Single-Molecule Localization 
Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (∼10–20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules. In this single-molecule based imaging approach, a wide variety of probes have proved valuable, ranging from genetically encodable photoactivatable fluorescent proteins to photoswitchable cyanine dyes. These have been used in diverse applications of superresolution imaging: from three-dimensional, multicolor molecule localization to tracking of nanometric structures and molecules in living cells. Single-molecule-based superresolution imaging thus offers exciting possibilities for obtaining molecular-scale information on biological events occurring at variable timescales.
PMCID: PMC3658623  PMID: 20055680
superresolution microscopy; single molecule; PALM; STORM; FPALM; diffraction limit; photoactivation; photoactivatable fluorescent protein; fluorescence imaging
10.  [No title available] 
PMCID: PMC3349341  PMID: 22242730
11.  Lessons in Fluctuation Correlation Spectroscopy 
Molecular diffusion and transport processes are fundamental in physical, chemical, and biological systems. Current approaches to measuring molecular transport in cells and tissues based on perturbation methods, e.g., fluorescence recovery after photobleaching, are invasive; single-point fluctuation correlation methods are local; and single-particle tracking requires the observation of isolated particles for relatively long periods of time. We discuss here the detection of molecular transport by exploiting spatiotemporal correlations measured among points at large distances (>1 μm). We illustrate the evolution of the conceptual framework that started with single-point fluorescence fluctuation analysis based on the transit of fluorescent molecules through a small volume of illumination. This idea has evolved to include the measurement of fluctuations at many locations in the sample using microscopy imaging methods. Image fluctuation analysis has become a rich and powerful technique that can be used to extract information about the spatial distribution of molecular concentration and transport in cells and tissues.
PMCID: PMC3576135  PMID: 21219151
pair correlation; molecular flow; anisotropic diffusion
12.  Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy 
The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy, including stimulated Raman scattering and two photon absorption, and pump-probe microscopy, including stimulated emission, excited state absorption and ground state depletion, provide distinct and powerful image contrasts for non-fluorescent species. Thanks to high-frequency modulation transfer scheme, they exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles, excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques.
PMCID: PMC3427791  PMID: 21453061
13.  Active Biological Materials 
Cells make use of dynamic internal structures to control shape and create movement. By consuming energy to assemble into highly organized systems of interacting parts, these structures can generate force and resist compression, as well as adaptively change in response to their environment. Recent progress in reconstituting cytoskeletal structures in vitro has provided an opportunity to characterize the mechanics and dynamics of filament networks formed from purified proteins. Results indicate that a complex interplay between length scales and timescales underlies the mechanical responses of these systems and that energy consumption, as manifested in molecular motor activity and cytoskeletal filament growth, can drive transitions between distinct material states. This review discusses the basic characteristics of these active biological materials that set them apart from conventional materials and that create a rich array of unique behaviors.
PMCID: PMC3236678  PMID: 18999991
cell mechanics; actin cytoskeleton; in vitro reconstitution; crawling motility; filopodia
14.  Role of Solvation Effects in Protein Denaturation: From Thermodynamics to Single Molecules and Back 
Protein stability often is studied in vitro through the use of urea and guanidinium chloride, chemical cosolvents that disrupt protein native structure. Much controversy still surrounds the underlying mechanism by which these molecules denature proteins. Here we review current thinking on various aspects of chemical denaturation. We begin by discussing classic models of protein folding and how the effects of denaturants may fit into this picture through their modulation of the collapse, or coil-globule transition, which typically precedes folding. Subsequently, we examine recent molecular dynamics simulations that have shed new light on the possible microscopic origins of the solvation effects brought on by denaturants. It seems likely that both denaturants operate by facilitating solvation of hydrophobic regions of proteins. Finally, we present recent single-molecule fluorescence studies of denatured proteins, the analysis of which corroborates the role of denaturants in shifting the equilibrium of the coil-globule transition.
PMCID: PMC3211090  PMID: 21219136
protein folding; urea; guanidinium chloride; hydrophobic effect; FRET; molecular dynamics simulations; single-molecule spectroscopy
15.  Solid State NMR Studies of Amyloid Fibril Structure 
Current interest in amyloid fibrils stems from their involvement in neurodegenerative and other diseases and from their role as an alternative structural state for many peptides and proteins. Solid state NMR methods have the unique capability of providing detailed structural constraints for amyloid fibrils, sufficient for the development of full molecular models. In this article, recent progress in the application of solid state NMR to fibrils associated with Alzheimer’s disease, prion fibrils, and related systems is reviewed, along with relevant developments in solid state NMR techniques and technology.
PMCID: PMC3191906  PMID: 21219138
Alzheimer’s disease; protein structure; prion; nuclear magnetic resonance
16.  Copper and the Prion Protein: Methods, Structures, Function, and Disease 
The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease.
PMCID: PMC2904554  PMID: 17076634
transmissible spongiform encephalopathies; electron paramagnetic resonance; octarepeat domain; neuroprotection; apoptosis
17.  Fluctuations in Biological and Bioinspired Electron-Transfer Reactions 
Central to theories of electron transfer (ET) is the idea that nuclear motion generates a transition state that enables electron flow to proceed, but nuclear motion also induces fluctuations in the donor-acceptor (DA) electronic coupling that is the rate-limiting parameter for nonadiabatic ET. The interplay between the DA energy gap and DA coupling fluctuations is particularly noteworthy in biological ET, where flexible protein and mobile water bridges take center stage. Here, we discuss the critical timescales at play for ET reactions in fluctuating media, highlighting issues of the Condon approximation, average medium versus fluctuation-controlled electron tunneling, gated and solvent relaxation controlled electron transfer, and the influence of inelastic tunneling on electronic coupling pathway interferences. Taken together, one may use this framework to establish principles to describe how macromolecular structure and structural fluctuations influence ET reactions. This framework deepens our understanding of ET chemistry in fluctuating media. Moreover, it provides a unifying perspective for biophysical charge-transfer processes and helps to frame new questions associated with energy harvesting and transduction in fluctuating media.
PMCID: PMC2883780  PMID: 20192814
coupling fluctuations; tunneling pathways; inelastic tunneling; pathway coherence; solvent dynamical effects; water-mediated tunneling
18.  Biological Cluster Mass Spectrometry 
This article reviews the new physics and new applications of secondary ion mass spectrometry using cluster ion probes. These probes, particularly C60, exhibit enhanced molecular desorption with improved sensitivity owing to the unique nature of the energy-deposition process. In addition, these projectiles are capable of eroding molecular solids while retaining the molecular specificity of mass spectrometry. When the beams are microfocused to a spot on the sample, bioimaging experiments in two and three dimensions are feasible. We describe emerging theoretical models that allow the energy-deposition process to be understood on an atomic and molecular basis. Moreover, experiments on model systems are described that allow protocols for imaging on biological materials to be implemented. Finally, we present recent applications of imaging to biological tissue and single cells to illustrate the future directions of this methodology.
PMCID: PMC2859288  PMID: 20055679
secondary ion mass spectrometry; bioimaging; molecular depth profiling; three-dimensional molecular imaging; C60; molecular dynamics
19.  Highly Fluorescent Noble Metal Quantum Dots 
Highly fluorescent, water-soluble, few-atom noble metal quantum dots have been created that behave as multi-electron artificial atoms with discrete, size-tunable electronic transitions throughout the visible and near IR. These “molecular metals” exhibit highly polarizable transitions and scale in size according to the simple relation, Efermi/N1/3, predicted by the free electron model of metallic behavior. This simple scaling indicates that fluorescence arises from intraband transitions of free electrons and that these conduction electron transitions are the low number limit of the plasmon – the collective dipole oscillations occurring when a continuous density of states is reached. Providing the “missing link” between atomic and nanoparticle behavior in noble metals, these emissive, water-soluble Au nanoclusters open new opportunities for biological labels, energy transfer pairs, and light emitting sources in nanoscale optoelectronics.
PMCID: PMC2735021  PMID: 17105412
gold cluster fluorescence; biolabels; jellium model; free electron scaling; single molecule microscopy

Results 1-19 (19)