Sub-angstrom conformational changes of a single molecule captured by AFM variance analysis

A system's equilibrium variance can be analyzed to probe its underlying dynamics at higher resolution. Here, using single-molecule atomic-force microscope techniques, we show how the variance in the length of a single dextran molecule can be used to establish thermodynamic equilibrium and to detect conformational changes not directly observable with other methods. Dextran is comprised of a chain of pyranose rings that each undergoes an Angstrom-scale transition from a chair to boat conformation under a stretching force. Our analysis of the variance of the molecule's fluctuations verifies equilibrium throughout the force-extension curve, consistent with the expected thermodynamic ensemble. This validates further analysis of the variance in the transition region, which reveals an intermediate conformation between the chair and the boat on the sub-Angstrom scale. Our test of thermal equilibrium as well as our variance analysis can be readily extended to a wide variety of molecules, including proteins.     

Molecular dynamics simulation of dextran extension by constant force in single molecule AFM

The extension of 1-6 polysaccharides has been studied in a series of recent single molecule AFM experiments. For dextran, a key finding was the existence of a plateau in the force-extension curve at forces between 700 and 1000 pN. We studied the extension of the dextran 10-mer under constant force using atomistic simulation with various force fields. All the force fields reproduce the experimental plateau on the force-extension curve. With AMBER94 and AMBER-GLYCAM04 force fields the plateau can be explained by a transition of the glucopyranose rings in the dextran monomers from the chair ((4)C(1)) to the inverted chair ((1)C(4)) conformation while other processes occur at smaller (rotation around C5-C6 bond) or higher (chairs to boat transitions) forces. The CHARMM force field provides a different picture which associates the occurrence of the plateau to chair-boat transitions of the glucopyranose rings.     

Twisting and untwisting a single DNA molecule covered by RecA protein

We study dsDNA-RecA interactions by exerting forces in the pN range on single DNA molecules while the interstrand topological state is controlled owing to a magnetic tweezers setup. We show that unwinding a duplex DNA molecule induces RecA polymerization even at moderate force. Once initial polymerization has nucleated, the extent of RecA coverage still depends on the degree of supercoiling: exerting a positive or negative torsional constraint on the fiber forces partial depolymerization, with a strikingly greater stability when ATPgammaS is used as a cofactor instead of ATP. This nucleofilament's sensitivity to topology might be a way for the bacterial cell to limit consumption of precious RecA monomers when DNA damage is addressed through homologous recombination repair.     

Allele-specific inhibition of divergent protein tyrosine phosphatases with a single small molecule

A central challenge of chemical biology is the development of small-molecule tools for controlling protein activity in a target-specific manner. Such tools are particularly useful if they can be systematically applied to the members of large protein families. Here we report that protein tyrosine phosphatases can be systematically 'sensitized' to target-specific inhibition by a cell-permeable small molecule, Fluorescein Arsenical Hairpin Binder (FlAsH), which does not inhibit any wild-type PTP investigated to date. We show that insertion of a FlAsH-binding peptide at a conserved position in the PTP catalytic-domain's WPD loop confers novel FlAsH sensitivity upon divergent PTPs. The position of the sensitizing insertion is readily identifiable from primary-sequence alignments, and we have generated FlAsH-sensitive mutants for seven different classical PTPs from six distinct subfamilies of receptor and non-receptor PTPs, including one phosphatase (PTP-PEST) whose three-dimensional catalytic-domain structure is not known. In all cases, FlAsH-mediated PTP inhibition was target specific and potent, with inhibition constants for the seven sensitized PTPs ranging from 17 to 370 nM. Our results suggest that a substantial fraction of the PTP superfamily will be likewise sensitizable to allele-specific inhibition; FlAsH-based PTP targeting thus potentially provides a rapid, general means for selectively targeting PTP activity in cell-culture- or model-organism-based signaling studies.     

The measurement of protein turnover by density labelling

A method for measuring the rate of protein degradation in plant tissue is described. The method uses density labelling to avoid difficulties associated with compartmentation and recycling of amino acids. Although the technique cannot be readily adapted to measure the rate of degradation of single proteins, it avoids difficulties of interpretation due to enzyme activation or inactivation. Values for the half-life of Lemna minor protein obtained by this method are compared with values obtained by a number of other methods. To obtain satisfactory results it was necessary to improve the method of isopycnic centrifugation in CsCl gradients. A considerable improvement was achieved by using KBr gradients, and the advantages of using KBr rather than CsCl for the separation of density-labelled protein are discussed.     

Nonequilibrium single molecule protein folding in a coaxial mixer

We have developed a continuous-flow mixing device suitable for monitoring bioconformational reactions at the single-molecule level with a response time of ∼10 ms under single-molecule flow conditions. Its coaxial geometry allows three-dimensional hydrodynamic focusing of sample fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. The capillary-based design enables rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each device to many months. In this article, to determine both the distance-to-time transfer function and the instrument response function of the device we characterize its fluid flow and mixing properties using both fluorescence cross-correlation spectroscopy velocimetry and finite element fluid dynamics simulations. We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By transiently populating the unfolded state of Chymotrypsin Inhibitor Protein 2 (CI2) under nonequilibrium in vitro refolding conditions, we spatially and temporally resolve the denaturant-dependent nonspecific collapse of the unfolded state from the barrier-limited folding transition of CI2. Our results are consistent with previous CI2 mixing results that found evidence for a heterogeneous unfolded state consisting of cis- and trans-proline conformers.     

The observation of structural transitions of a single protein molecule

Coherent neutron scattering measurements of an amorphous, in vivo deuterated C-phycocyanin are compared with a calculation of the individual protein molecule's coherent static structure factor. Both show the significant features associated with known structure factors of several amorphous materials, most notably, an unusually sharp first diffraction peak occurring near 1.4 A(-1). We show that in the protein, such a peak results from the product of a form factor associated with correlations of atoms within individual amino acids and a structural term expressing inter-amino-acid correlations. The measurement, interpreted through behavior of the first diffraction peak, indicates that inter-amino-acid correlations - a measure of the protein's medium-range structure - undergo transitions which are primarily related to hydration rather than to temperature.     

Spectral fluctuation of a single fluorophore conjugated to a protein molecule

We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous buffer to methanol in both single-molecule and bulk measurements were similar, indicating that, even at the single molecule level, the fluorescence spectrum is sensitive to microenvironmental changes of fluorophores. The time dependence of the fluorescence spectra of fluorophores attached to S1 molecules solely showed a fluctuation with time over a time scale of seconds. Because the fluorescence spectra of the same fluorophores directly conjugated to a glass surface remained constant, the spectral fluctuation observed for the fluorophores attached to S1 is most likely due to slow spontaneous conformational changes in the S1 molecule. Thus, single-molecule fluorescence spectroscopy appears to be a powerful tool to study the dynamic behavior of single biomolecules.     

Plasma very low density lipoproteins contain a single molecule of apolipoprotein B

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.     

Probing the kinetics of single molecule protein folding

We propose an approach to integrate the theory, simulations, and experiments in protein-folding kinetics. This is realized by measuring the mean and high-order moments of the first-passage time and its associated distribution. The full kinetics is revealed in the current theoretical framework through these measurements. In the experiments, information about the statistical properties of first-passage times can be obtained from the kinetic folding trajectories of single molecule experiments (for example, fluorescence). Theoretical/simulation and experimental approaches can be directly related. We study in particular the temperature-varying kinetics to probe the underlying structure of the folding energy landscape. At high temperatures, exponential kinetics is observed; there are multiple parallel kinetic paths leading to the native state. At intermediate temperatures, nonexponential kinetics appears, revealing the nature of the distribution of local traps on the landscape and, as a result, discrete kinetic paths emerge. At very low temperatures, exponential kinetics is again observed; the dynamics on the underlying landscape is dominated by a single barrier. The ratio between first-passage-time moments is proposed to be a good variable to quantitatively probe these kinetic changes. The temperature-dependent kinetics is consistent with the strange kinetics found in folding dynamics experiments. The potential applications of the current results to single-molecule protein folding are discussed.     

Surface charge density estimation by 9-aminoacridine fluorescence titration: improvements and limitations

A large number of surface charge density () and surface potential (_o) estimations have been based on 1) titrations of the fluorescence of 9-aminoacridine released from the diffuse double layer adjacent to negatively charged membrane surfaces by non-adsorbing monovalent and divalent cations, and 2) calculations using experimental data from the titration curves and the Gouy-Chapman theory of the diffuse double layer. In this paper we discuss the different simplifying approximations employed in the earlier calculations and recommend modified formulas for the calculations. The latter have been derived without any simplifying approximation concerning the ionic (electrolyte) composition of the titration assays. We also show that depends, to some extent, on the concentrations of buffer and vesicles in the assays and present experimental evidence that decamethonium (decane-1,10-bis-trimethylammonium), a bulky organic divalent cation, can be satisfactorily used for the estimation of under well-defined conditions, despite its putative interaction with membranes.Abbreviations 9-AA 9-aminoacridine - (DeM)²⁺ decamethonium - (DiM)²⁺ dimethonium - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glyol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - (HeM)²⁺ hexamethonium - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 4-morpholinopropanesulfonic acid - PM plasma membrane - Tris tris(hydroxymethyl)aminomethane - surface charge density - _o surface potential Correspondence to: A. Bérczi     

Reaction of lectins with human erythrocytes : III. Surface charge density and agglutination

A number of commercially available enzymes were used to modify the cell surface of human erythrocytes to varying degrees. In protease-treated erythrocytes the decrease in surface charge (determined by cell electrophoresis or analysis of sialic acid content) correlates with an increase in agglutinability with concanavalin A (ConA) and wheat germ agglutinin (WGA). On the other hand, treatment with neuraminidase leads to very large decrease in surface charge with only an intermediate increase in agglutinability with both lectins. Subsequent protease treatment of these cells enhances their agglutinability appreciably without further altering their surface charge. It is concluded that the increased agglutinability following protease treatment is due both to a decrease in the net negative charge and a removal of peptides and glycopeptides from the cell surface that may sterically hinder the agglutination reaction.     

Direct measurement of DNA by means of AFM.

We have demonstrated that the electrostatic stretch-and-positioning method is useful for the analysis of a long DNA molecule by means of atomic force microscopy (AFM). DNA molecules were stretched parallel to the field line, and immobilized onto the aluminum electrodes patterned on a glass plate. Through AFM observation, we confirmed the immobilization of individual DNA molecules, not aggregate.     

Assembly of membrane-bound protein complexes: detection and analysis by single molecule diffusion

Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus, a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca(2+)-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves, identifies and quantitates the rapidly diffusing monomers and slowly diffusing heterodimers. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing ratio of monomers and heterodimers, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ~10(2)-fold greater in the membrane system than in solution, apparently due to both faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.     

Viscoelastic study of the mechanical unfolding of a protein by AFM

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.     

Surface charge density determines the efficiency of cationic gemini surfactant based lipofection

The efficiencies of the binary liposomes composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and cationic gemini surfactant, (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide as transfection vectors, were measured using the enhanced green fluorescent protein coding plasmid and COS-1 cells. Strong correlation between the transfection efficiency and lipid stoichiometry was observed. Accordingly, liposomes with X(SR-1) > or = 0.50 conveyed the enhanced green fluorescent protein coding plasmid effectively into cells. The condensation of DNA by liposomes with X(SR-1) > 0.50 was indicated by static light scattering and ethidium bromide intercalation assay, whereas differential scanning calorimetry and fluorescence anisotropy of diphenylhexatriene revealed stoichiometry dependent reorganization in the headgroup region of the liposome bilayer, in alignment with our previous Langmuir-balance study. Surface charge density and the organization of positive charges appear to determine the mode of interaction of DNA with (2S,3R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide/1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes, only resulting in DNA condensation when X(SR-1) > 0.50. Condensation of DNA in turn seems to be required for efficient transfection.