High-Bandwidth Protein Analysis Using Solid-State Nanopores

High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D₀) by a factor of ∼50, and are voltage-independent in the regime tested. We reason that deviations of D from D₀ are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.     

Protein Mobility in the Cytoplasm of Escherichia coli

The rate of protein diffusion in bacterial cytoplasm may constrain a variety of cellular functions and limit the rates of many biochemical reactions in vivo. In this paper, we report noninvasive measurements of the apparent diffusion coefficient of green fluorescent protein (GFP) in the cytoplasm of Escherichia coli. These measurements were made in two ways: by photobleaching of GFP fluorescence and by photoactivation of a red-emitting fluorescent state of GFP (M. B. Elowitz, M. G. Surette, P. E. Wolf, J. Stock, and S. Leibler, Curr. Biol. 7:809–812, 1997). The apparent diffusion coefficient, D_a, of GFP in E. coli DH5α was found to be 7.7 ± 2.5 μm²/s. A 72-kDa fusion protein composed of GFP and a cytoplasmically localized maltose binding protein domain moves more slowly, with D_a of 2.5 ± 0.6 μm²/s. In addition, GFP mobility can depend strongly on at least two factors: first, D_a is reduced to 3.6 ± 0.7 μm²/s at high levels of GFP expression; second, the addition to GFP of a small tag consisting of six histidine residues reduces D_a to 4.0 ± 2.0 μm²/s. Thus, a single effective cytoplasmic viscosity cannot explain all values of D_a reported here. These measurements have implications for the understanding of intracellular biochemical networks.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Dynamic Light-Scattering Evidence for the Flexibility of Native Muscle Thin Filaments

We have obtained clear evidence for the flexibility of native scallop adductor thin filaments by studying the temperature and ionic strength dependence of the average decay constants obtained from intensity fluctuation spectroscopic (IFS) measurements. The low-angle (10-25°), average decay constants obtained from time autocorrelation functions of scattered light were independent of concentration (0.08-1.3 mg/ml), scaled with the ratio of temperature to solvent viscosity, T/η, over a range of 4-45°C, and yielded a value for the translational diffusion coefficient of D_T^5°C = (1.24 ± 0.06) × 10^-8 cm²/s. From this value and the Broersma relation for rigid rods, we find an average filament length of 1.06 ± 0.06 μm. Quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that at high temperatures (> 35°C) or in 0.6 M NaCl, tropomyosin completely dissociates from native thin filaments. Decay constants from high-angle (60-150°C) IFS temperature dependence measurements do not scale with T/η and hence do not show the temperature dependence expected for rigid rods. The differences are not due to any change in length distribution of filaments with temperature or to the free tropomyosin in solution, but are attributed to nonrigid motions of the filaments. Similar experiments on samples in high- and low-salt solvents gave results consistent with this interpretation.     

Helicobacter pylori Type IV Secretion Apparatus Exploits β1 Integrin in a Novel RGD-Independent Manner

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with α5β1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell β1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to α5β1 integrin is rather strong (dissociation constant, K_D of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (K_D of 15 nM). For CagA translocation the extracellular part of the β1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of β1 integrin-specific monoclonal antibodies directed against various defined β1 integrin epitopes, such as the PSI, the I-like, the EGF or the β-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of β1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the β1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation.     

The voltage-dependent step of the chloride transporter of Valonia utricularis encounters a Nernst-Planck and not an Eyring type of potential energy barrier

Voltage-clamp experiments were performed on cells of the giant marine alga Valonia utricularis to study the voltage dependence of the previously postulated chloride transporter (Wang, J., G. Wehner, R. Benz, and U. Zimmermann. 1991. Biophys. J. 59:235-248). Only one exponential current relaxation (apart from the capacitive spike) could be resolved up to a clamp voltage of ~120 mV within the time resolution of our experimental instrumentation (100 μs). This means that the rate constants of the heterogeneous complexation, k_R (association) and k_D (dissociation), were too fast to be resolved. Therefore, the “Läuger” model for carrier-mediated ion transport with equilibrium heterogeneous surface reaction was used to fit the experimental results. The voltage dependence of the initial membrane conductance was used for the evaluation of the voltage dependence of the translocation rate constant of the complexed carriers, k_AS. The initial conductance was found to be independent on the clamp voltage, which means that the translocation rate constant k_AS is a linear function of the applied voltage and that the voltage dependence of the translocation of charged carriers through the plasmalemma could be explained by a square-type Nernst-Planck barrier. The movement of the complexed form of the carrier through the membrane may be explained by a diffusion process rather than by simple first-order kinetic jump across an Eyring-type potential well. The current relaxation after a voltage clamp was studied as a function of the external chloride concentration. The results allowed an estimation of the stability constant, K, of the heterogeneous complexation reaction and a calculation of the translocation rate constants of the free and the complexed carriers, k_s and k_AS, respectively.     

Distributed Kinetics of the Charge Movements in Bacteriorhodopsin: Evidence for Conformational Substates

The flash-induced charge movements during the photocycle of light-adapted bacteriorhodopsin in purple membranes attached to a black lipid membrane were investigated under voltage clamp and current clamp conditions. Signal registration ranged from 200 ns to 30 s after flash excitation using a logarithmic clock, allowing the equally weighted measurement of the electrical phenomena over eight decades of time. The active pumping signals were separated from the passive system discharge on the basis of an equivalent circuit analysis. Both measuring methods were shown to yield equivalent results, but the charge translocation could be accurately monitored over the whole time range only under current clamp conditions. To describe the time course of the photovoltage signals a model based on distributed kinetics was found to be more appropriate than discrete first order processes suggesting the existence of conformational substates with distributed activation energies. The time course of the active charge displacement is characterised by a continuous relaxation time spectrum with three broad peaks plus an unresolved fast transient (<0.3 μs) of opposite polarity. The time constants and relative amplitudes (in brackets) derived from the peak rate constants and relative areas of the three bands are: τ₁ = 32 μs (20%), τ₂ = 0.89 ms (15%) and τ₃ = 18 ms (65%) at 25°C in 150 mM KCl at pH7. The Arrhenius plots of the peak rate constants were linear yielding activation energies of E_A1 = 57 kJ/mol, E_A2 = 52 kJ/mol, and E_A3 = 44 kJ/mol. The electrical signal at 890 μs has no counterpart in the photocycle of bacteriorhodopsin suspensions. Fits with a sum of exponentials required 5 to 6 components and were not reproducible. Analysis of photoelectrical signals with continuous relaxation time spectra gave equally good fits with fewer parameters and were well reproducible.     

Diffusion of Single Cardiac Ryanodine Receptors in Lipid Bilayers Is Decreased by Annexin 12

Diffusion of cardiac ryanodine receptors (RyR2) in lipid bilayers was characterized. RyR2 location was monitored by imaging fluo-3 fluorescence due to Ca²⁺ flux through RyR2 channels or fluorescence from RyR2 conjugated with Alexa 488 or containing green fluorescent protein. Single channel currents were recorded to ensure that functional channels were studied. RyR2 exhibited an apparent diffusion coefficient (D_RyR) of 1.2 × 10⁻⁸ cm² s⁻¹ and a mean path length of 5.0 μm. Optimal use of optical methods for analysis of RyR2 channel function requires that RyR2 diffusion be limited. Therefore, we tested the effect of annexin 12, which interacts with anionic phospholipids in a Ca²⁺-dependent manner. Addition of annexin 12 (0.25–4.0 μM) to the trans side of bilayers containing an 80:20 ratio of phosphatidylethanolamine/phosphatidylserine decreased RyR2 diffusion in a concentration-dependent manner. Annexin 12 (2 μM) decreased the apparent D_RyR 683-fold from 1.2–10⁻⁸ to 1.8 × 10⁻¹¹ cm² s⁻¹ and the mean path length 10-fold from 5.0 to 0.5 μm without obvious changes in the conductance of the native bilayer or in activation of RyR2 channels by Ca²⁺ or suramin. Thus, annexin 12 may provide a useful tool for optimizing optical analysis of RyR2 channels in lipid bilayers.     

Quantifying Interactions of a Membrane Protein Embedded in a Lipid Nanodisc using Fluorescence Correlation Spectroscopy

Using fluorescence correlation spectroscopy, we measured a dissociation constant of 20 nM between EGFP-labeled LcrV from Yersinia pestis and its cognate membrane-bound protein YopB inserted into a lipid nanodisc. The combination of fluorescence correlation spectroscopy and nanodisc technologies provides a powerful approach to accurately measure binding constants of interactions between membrane bound and soluble proteins in solution. Straightforward sample preparation, acquisition, and analysis procedures make this combined technology attractive for accurately measuring binding kinetics for this important class of protein-protein interactions.Interactions involving membrane proteins are integral to a multitude of cellular processes, including signal transduction, energy production and conversion, cell adhesion, and foreign molecule identification. More than half of all pharmaceutical drugs target membrane proteins, further illustrating their importance in human health (1). Due to this, there is a continuing high demand for methods that can screen, validate, and quantify interactions involving membrane proteins. Unfortunately, the quantitative techniques available to characterize protein-protein interactions are most often directed toward soluble proteins, and are often difficult to apply to membrane proteins (2). Recently, advances in lipid nanodisc technologies, often referred to as reconstituted high-density lipoproteins, have enabled biophysical and biochemical studies of solubilized membrane proteins. The nativelike lipid environment of nanodiscs maintains protein functionality, opening a realm of possibilities in analyzing membrane protein function and dynamics in solution (3).Nanodiscs are discoidal cell membrane mimetics that are 8–20 nm in diameter, consisting of a lipid bilayer stabilized by two peripheral apolipoprotein A-I proteins (4). These particles provide an excellent alternative to traditional lipid-based platforms (e.g., liposomes) for membrane protein solubilization and interrogation. The diameter of nanodisc can be engineered to accommodate different-sized membrane proteins by varying lipid composition. As such, nanodiscs represent an important platform for expression, isolation, and study of functional membrane proteins and the multiprotein complexes they form. Several groups have successfully reconstituted a variety of membrane proteins, including bacteriorhodopsin (5), G-protein coupled receptors (6), and cytochrome P450 (7) to name a few. To produce solubilized, discrete membrane proteins, we utilized a cell-free expression approach to embed membrane proteins directly into nanodiscs formed in situ. This approach allows for enhanced purification and rapid labeling of proteins of interest where traditional approaches are unsuccessful (8). Fluorescence correlation spectroscopy (FCS) analysis coupled with nanodisc technology has been successfully used to monitor small ligand binding interactions with membrane proteins (6,8) and to measure lipid-protein interactions at the single molecule level (9). Here, we show that FCS can be used to measure interactions between soluble proteins and cognate membrane proteins inserted into nanodiscs produced using cell-free expression methodologies.FCS uses correlation analysis of fluorescence arising from randomly diffusing molecules to measure diffusion constants, and hence hydrodynamic radii, of species in solution. Diffusion times measured by FCS are inversely proportional to diffusion constants. A shift to slower diffusion time indicates an increase in the hydrodynamic radius due to binding. Due to this size sensitivity, FCS has been proposed repeatedly as a method to quantify molecular interactions in solution (10). However, an important difficulty in FCS arises when characterizing interactions between two proteins of similar size. The diffusion time is inversely proportional to the cube root of the molecular mass τ_D ∼ M^−1/3. Doubling the mass results in only a factor-of-1.3 increase in diffusion time, but a factor of 1.6 is required to resolve two species (11). As we demonstrate, this difficulty is overcome by using FCS to measure the interactions between labeled soluble proteins and membrane proteins supported within a nanodisc. The much larger size of the membrane protein-nanodisc complex (Fig. 1 A) relative to most soluble proteins provides the necessary differential in diffusion times to easily resolve bound and free species by FCS.Open in a separate windowFigure 1(A) Model of YopB (blue) inserted into a 10-nm nanodisc with cognate protein LcrV (red) labeled with EGFP (green). The molecular masses used in this model are: LcrV (35 kDa), EGFP (27 kDa), YopB (42 kDa, monomer), and nanodisc-YopB complex (258 kDa). The small LcrV binds to the much larger YopB-nanodisc complex, resulting in a significant shift in the autocorrelation curves to longer diffusion times. (B) Hypothetical autocorrelation curves expected for a series of titration experiments measured using FCS. Increased binding is measured as an increase in diffusion time. For intermediate binding, diffusion components are detectable as illustrated (red, green, and blue curves). To see this figure in color, go online.Binding kinetics are readily measured using FCS by titrating increasing amounts of unlabeled cognate protein, resulting in a series of autocorrelation curves similar to those modeled in a hypothetical example in Fig. 1 B. For 0% binding (black curve), only the labeled soluble protein is present, producing a single component correlation curve with a diffusion time of 0.1 ms. On the autocorrelation curve with a single component, the diffusion time is the time at which the autocorrelation amplitude decreases by half. For 100% binding (cyan curve), the amount of nanodisc with inserted membrane protein is well above the dissociation constant so that all available soluble protein is bound.Again, a single component correlation curve is found, but now with a longer diffusion time (10 ms in this hypothetical example). Intermediate cases have two components, corresponding to free and bound protein. The relative amplitudes of the components obtained by fitting to two component models in these cases can be used to obtain a binding curve. For binding interactions, the amount of ligand in the free and bound state can be separated by their diffusion time and quantified as a function of concentration. The fraction of bound protein is calculated by fitting the correlation to two components. The first component f₀(τ) is the correlation of the free protein. The second component g₀(τ) is the correlation when the protein is bound. The total correlation function ish(τ) = αf₀(τ) + βg₀(τ), (1)where f₀(τ) = 1/(1 + τ/τ_f) and g₀(τ) = 1/(1 + τ/τ_b). The values α and β are, respectively, the amplitudes of the correlation function for free and bound protein, with the corresponding diffusion times τ_f and τ_b. The ratio F = β/(α + β) gives the fraction bound.We investigated by FCS the interaction of two Yersinia pestis proteins: soluble EGFP-labeled LcrV and membrane-bound YopB (Fig. 1 A). YopB is an effector protein involved in host cell invasion and disarming the cell’s defense. Although the structure of YopB is largely unknown, it is thought to exist as a dimer (12). Delivery of YopB to the host cell is regulated by the Type III secretion system (T3SS) (i.e., injectisome). The needle tip of the injectisome contains the LcrV protein. When Y. pestis comes in contact with a cell membrane, the injectisome forms a pore through the membrane to facilitate the diffusion of effector proteins into the host cell. Multiple lines of evidence suggest that pores are formed only through the direct interaction of YopB with LcrV (13).The FCS diffusion times of free LcrV (τ_f; Fig. 2 A, black curve) and fully bound LcrV (τ_b; [YopB] = 10 μM) were found to be 140 and 630 μs, respectively. Because no measurable change in diffusion time was detected above [YopB] = 1 μM (Fig. 2, green curve), LcrV was assumed to be completely bound at [YopB] = 10 μM. The diffusion times of free and bound LcrV were determined by fitting the autocorrelation curves to a one-component model (χ² ∼ 1 for each diffusion time). These diffusion times correspond to average hydrodynamic radii of 2.5 nm for free LcrV and 11.1 nm for the LcrV + YopB-nanodisc complex, as calculated by the Einstein-Stokes equation. No significant change in diffusion time was detected when LcrV was incubated with 2 μM of nanodiscs lacking the YopB protein (Fig. 2 A, gray curve), suggesting that LcrV alone is unable to interact directly with a lipid bilayer.Open in a separate windowFigure 2(A) FCS autocorrelation curves of 1 nM EGFP-labeled LcrV protein in the absence and presence of YopB at increasing concentrations. The diffusion time of freely diffusing LcrV is τ_D,free = 140 s (black curve). At [YopB] = 1 μM where LcrV is completely bound, the FCS curve is dominated by a single component (green curve). The autocorrelation curves at intermediate concentrations (in particular [YopB] = 25 and 50 nM, blue and cyan curves) contain contributions from two diffusing components. A control experiment was performed with 2 μM of nanodisc lacking the YopB protein (gray curve). No significant change in diffusion time was detected. (B) The fraction of bound LcrV as a function of YopB concentration (log scale). This fraction is calculated by fitting the FCS curves shown in Fig. 2A with Eq. 1. The dissociation constant extracted is K_d = 20.45 ± 2.0 nM. Error bars represent an average over six FCS measurements. To see this figure in color, go online.To extract the dissociation constant (K_D), a titration was performed over a range of YopB concentrations (10 pM to 1 μM), obtained by varying the concentration of YopB-nanodisc complexed. Having determined the values of the diffusion times for free (τ_f) and bound (τ_b) protein, we fitted the autocorrelation curves to a two-species correlation function with Eq. 1. The brightness per LcrV molecule was between 2170 and 2280 photons/(s × molecule) for the entire titration, validating the use of Eq. 1. The only varying parameters were the correlation amplitudes α (free protein) and β (bound protein). Intermediate binding was observed between 10 and 100 nM, because two correlation components are resolved. At [YopB] = 50 nM (cyan curve), the ratio β/α was 1.5, indicating that more than half of LcrV was bound. For the two-component model in these intermediate cases, the χ² value was between 1 and 1.3, indicating a good fit. In contrast, fitting the data to a one-component model resulted in a poor fit (χ² > 7), justifying the use of the two-species model. Binding curves were fitted to the equation y = x/(K_D + x), where y is the fraction of bound LcrV, x is the YopB concentration, and K_d the dissociation constant.Fitting this model with a least-squares algorithm (Fig. 2 B), we extracted K_d = 20.5 ± 2.0 nM. To quantify the statistical error, three measurements were recorded for 2 min, and the entire titration was repeated. Importantly, these data provide not only a quantitative binding affinity for the YopB-LcrV interaction, but support the hypothesis that LcrV requires direct interaction with YopB, not just a lipid bilayer, to promote pore formation.In conclusion, the coupling of FCS with nanodisc technology provides a facile yet powerful tool to quantitatively measure interactions involving membrane proteins in solution. FCS can simultaneously detect the presence of both free and bound species without the need for surface immobilization of the cognate proteins. Cell-free coexpression of both membrane protein and apolipoprotein components in the presence of lipids is a facile methodology for producing functional, soluble, nanodisc-supported membrane proteins. Labeling the soluble cognate proteins with EGFP provides a simple path to obtaining fluorescent, single-labeled proteins compatible with FCS. The combination of FCS with nanodisc technology presented here provides not only new key data for modeling the invasion process of Y. pestis, but can also be generalized to study interactions between most other soluble and membrane proteins. Such methods have been lacking, yet are critical for understanding interaction networks, e.g., signal transduction cascades.See the Supporting Material for additional detail on the methodology used.     

Response Characteristics of Soil Fractal Features to Different Land Uses in Typical Purple Soil Watershed

As a fundamental characteristic of soil physical properties, the soil Particle Size Distribution (PSD) is important in the research on soil moisture migration, solution transformation, and soil erosion. In this research, the PSD characteristics with distinct methods in different land uses are analyzed. The results show that the upper bound of the volume domain of the clay domain ranges from 5.743μm to 5.749μm for all land-use types. For the silt domain of purple soil, the value ranges among 286.852~286.966 μm. For all purple soil land-use types, the order of the volume domain fractal dimensions is D_clay<D_silt<D_sand. However, the values of D_silt and D_sand in the Pinus massoniana Lamb, Robinia pseudoacacia L and Ipomoea batatas are all higher than the corresponding values in the Citrus reticulate Blanco and Setaria viridis. Moreover, in all the land-use types, all of the parameters in volume domain fractal dimension (D_vi) are higher than the corresponding parameter values from the United States Department of Agriculture (D_vi(U)). The correlation study between the volume domain fractal dimension and the soil properties shows that the intensity of correlation to the soil texture and soil organic matter has the order as: D_silt>D_silt(U)>D_sand (U)>D_sand and D_silt>D_silt(U)>D_sand>D_sand(U), respectively. As it is compared with all D_vi, the D_silt has the most significant correlativity to the soil texture and organic matter in different land uses of the typical purple soil watersheds. Therefore, D_silt will be a potential indictor for evaluating the proportion of fine particles in the PSD, as well as a key measurement in soil quality and productivity studies.     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.     

Spatiotemporal dynamics of the COPI vesicle machinery

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D ≈ 15 μm² s⁻¹), whereas coatomer diffuses 5–10 times more slowly than expected (D ≈ 1 μm² s⁻¹). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.     

From microscopy data to in silico environments for in vivo-oriented simulations

In our previous study, we introduced a combination methodology of Fluorescence Correlation Spectroscopy (FCS) and Transmission Electron Microscopy (TEM), which is powerful to investigate the effect of intracellular environment to biochemical reaction processes. Now, we developed a reconstruction method of realistic simulation spaces based on our TEM images. Interactive raytracing visualization of this space allows the perception of the overall 3D structure, which is not directly accessible from 2D TEM images. Simulation results show that the diffusion in such generated structures strongly depends on image post-processing. Frayed structures corresponding to noisy images hinder the diffusion much stronger than smooth surfaces from denoised images. This means that the correct identification of noise or structure is significant to reconstruct appropriate reaction environment in silico in order to estimate realistic behaviors of reactants in vivo. Static structures lead to anomalous diffusion due to the partial confinement. In contrast, mobile crowding agents do not lead to anomalous diffusion at moderate crowding levels. By varying the mobility of these non-reactive obstacles (NRO), we estimated the relationship between NRO diffusion coefficient (D_nro) and the anomaly in the tracer diffusion (α). For D_nro=21.96 to 44.49 μm²/s, the simulation results match the anomaly obtained from FCS measurements. This range of the diffusion coefficient from simulations is compatible with the range of the diffusion coefficient of structural proteins in the cytoplasm. In addition, we investigated the relationship between the radius of NRO and anomalous diffusion coefficient of tracers by the comparison between different simulations. The radius of NRO has to be 58 nm when the polymer moves with the same diffusion speed as a reactant, which is close to the radius of functional protein complexes in a cell.     

Role of periplasmic trehalase in uptake of trehalose by the thermophilic bacterium Rhodothermus marinus

Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: K_m = 156 ± 11 μM and V_max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (K_m = 0.11 ± 0.03 μM and V_max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (K_m = 46 ± 3 μM and V_max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.     

Diffusion of insulin-like growth factor-I and ribonuclease through fibrin gels

A fluorescence-based method for simultaneously determining the diffusion coefficients of two proteins is described, and the diffusion coefficient of insulin-like growth factor (IGF-I) and ribonuclease (RNase) in a 0.27% fibrin hydrogel is reported. The method is based on two-color imaging of the relaxation of the protein concentration field with time and comparing the results with a transport model. The gel is confined in a thin (200 μm) capillary and the protein is labeled with a fluorescent dye. The experimentally determined diffusion coefficient of RNase (D = 1.21 × 10⁻⁶ cm²/s) agrees with literature values for dilute gels and bulk aqueous solutions, thus indicating the gel and the dye had a negligible effect on diffusion. The experimental diffusion coefficient of IGF-I (D = 1.59 × 10⁻⁶ cm²/s), in the absence of binding to the fibrin matrix, is consistent with the dimensions of the molecule known from x-ray crystallography and a correlation between D and molecular weight based on 14 other proteins. The experimental method developed here holds promise for determining molecular transport properties of biomolecules under a variety of conditions, for example, when the molecule adsorbs to the gel or is convected through the gel by fluid transport.     

Photochemical and Nonphotochemical Reactions of Phytochrome in vivo

The nonphotochemical reactions of phytochrome in the coleoptiles of dark-grown corn seedlings were studied at 3 temperatures: 14°, 24°, and 34°. The data obtained show that the destruction of P_fr is the only measurable reaction occurring; reversion of P_fr to P_r was not found. The Q₁₀'s (2.7 and 3.5) and zero order kinetics found for the destruction reaction are consistent with the hypothesis that the reaction is enzyme-mediated.

In vivo action spectra for phytochrome transformation in the coleoptiles of darkgrown corn seedlings were obtained which agree qualitatively with those obtained by other workers for phytochrome-mediated physiological responses and in vitro action spectra. In vivo conversion of phytochrome by blue light, as determined from spectrophotometric measurements of phytochrome itself, is reported. Action peaks for P_r were found at 667 mμ and in the blue in the region of 400 mμ, with a broad shoulder from 590 mμ to 640 mμ. Action peaks for P_fr were found at 725 mμ and in the blue in the region of 400 mμ with a minor peak at 670 mμ, and a broad shoulder from 590 mμ to 640 mμ. The ratio of the quantum efficiencies of P_r at 667 mμ and P_fr at 725 mμ (Φ_r667/Φ_fr725) was estimated to be 1.0.

     

Microstructural Analysis of Peripheral Lung Tissue through CPMG Inter-Echo Time R2 Dispersion

Since changes in lung microstructure are important indicators for (early stage) lung pathology, there is a need for quantifiable information of diagnostically challenging cases in a clinical setting, e.g. to evaluate early emphysematous changes in peripheral lung tissue. Considering alveoli as spherical air-spaces surrounded by a thin film of lung tissue allows deriving an expression for Carr-Purcell-Meiboom-Gill transverse relaxation rates R ₂ with a dependence on inter-echo time, local air-tissue volume fraction, diffusion coefficient and alveolar diameter, within a weak field approximation. The model relaxation rate exhibits the same hyperbolic tangent dependency as seen in the Luz-Meiboom model and limiting cases agree with Brooks et al. and Jensen et al. In addition, the model is tested against experimental data for passively deflated rat lungs: the resulting mean alveolar radius of R _A = 31.46 ± 13.15 μm is very close to the literature value (∼34 μm). Also, modeled radii obtained from relaxometer measurements of ageing hydrogel foam (that mimics peripheral lung tissue) are in good agreement with those obtained from μCT images of the same foam (mean relative error: 0.06 ± 0.01). The model’s ability to determine the alveolar radius and/or air volume fraction will be useful in quantifying peripheral lung microstructure.     

Early Picosecond Events in the Photocycle of Bacteriorhodopsin

The primary processes of the photochemical cycle of light-adapted bacteriorhodopsin (BR) were studied by various experimental techniques with a time resolution of 5 × 10^-13 s. The following results were obtained. (a) After optical excitation the first excited singlet state S₁ of bacteriorhodopsin is observed via its fluorescence and absorption properties. The population of the excited singlet state decays with a lifetime τ₁ of ~0.7 ps (430 ± 50 fs) (52). (b) With the same time constant the first ground-state intermediate J builds up. Its absorption spectrum is red-shifted relative to the spectrum of BR by ~30 nm. (c) The second photoproduct K, which appears with a time constant of τ₂ = 5 ps shows a red-shift of 20 nm, relative to the peak of BR. Its absorption remains constant for the observation time of 300 ps. (d) Upon suspending bacteriorhodopsin in D₂O and deuterating the retinal Schiff base at its nitrogen (lysine 216), the same photoproducts J and K are observed. The relaxation time constants τ₁ and τ₂ remain unchanged upon deuteration within the experimental accuracy of 20%.     

Cellular Microcystin Content in N-Limited Microcystis aeruginosa Can Be Predicted from Growth Rate

Cell quotas of microcystin (Q_MCYST; femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q_MCYST and specific growth rate (μ), from which we propose a generalized model that enables Q_MCYST at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q_MCYST from μ, μ_max (maximum specific growth rate), Q_MCYSTmax (maximum cell quota), and Q_MCYSTmin (minimum cell quota). Under the conditions examined in this study, we predict a Q_MCYSTmax of 0.129 fmol cell⁻¹ at μ_max and a Q_MCYSTmin of 0.050 fmol cell⁻¹ at μ = 0. Net MCYST production rate (R_MCYST) asymptotes to zero at μ = 0 and reaches a maximum of 0.155 fmol cell⁻¹ day⁻¹ at μ_max. MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with μ, whereas the MCYST/protein ratio reached a maximum at intermediate μ. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with μ, leading to an increase in intracellular MCYST concentration at high μ. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.     

Three Distinct Mechanisms for Translocation and Activation of the δ Subspecies of Protein Kinase C

We expressed δ subspecies of protein kinase C (δ-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The δ-PKC–GFP fusion protein had enzymological characteristics very similar to those of the native δ-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of δ-PKC–GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 μM), induced a slower translocation of δ-PKC–GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of δ-PKC–GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated δ-PKC–GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H₂O₂; 5 mM) failed to translocate δ-PKC–GFP but increased its kinase activity more than threefold. δ-PKC–GFP was strongly tyrosine phosphorylated when treated with H₂O₂ but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of δ-PKC–GFP even after treatment with H₂O₂. Simultaneous treatment with TPA and H₂O₂ further activated δ-PKC–GFP up to more than fivefold. TPA treatment of cells overexpressing δ-PKC–GFP led to an increase in the number of cells in G₂/M phase and of dikaryons, while stimulation with H₂O₂ increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of δ-PKC.