Neutrophil-bead collision assay: pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime

Visualization of flowing neutrophils colliding with adherent 1-mum-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (epsilon), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s(-1), epsilon decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, L(tether)(m), increased from 0.35 mum to approximately 2.0 mum over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress off-rate, k(off)(0) = 0.56 s(-1) and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (+/- tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3-5 (11%)). For collisions at 100 s(-1), pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on epsilon, but increased L(tether)(m) by 1.74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-beta-cyclodextrin or formaldehyde fixation had no effect on epsilon, but reduced L(tether)(m) by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophil-bead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes.     

Distribution of unselectively bound ligands along DNA

Unselective and reversible adsorption of ligands on DNA for a model of binding proposed by Zasedatelev, Gursky, and Volkenshtein is considered. In this model, the interaction between neighboring ligands located at the distance of i binding centers is characterized by the statistical weight a(i). Each ligand covers L binding centers. For this model, expressions for binding averages are represented in a new simple form. This representation is convenient for the calculation of the fraction of inter-ligand distances of i binding centers f(d)(i) and the fraction of binding centers included in the distances of i binding centers f(bc)(i) for various types of interaction between bound ligands. It is shown that, for non-cooperative binding, contact cooperativity and long-range cooperativity, the fraction of the zero inter-ligand distance f(d)(0) is maximal at any relative concentration of bound ligands (r). Calculations demonstrate that, at low r, f(d)(0) approximately r . a(o), and f(d)(i) approximately r at 1 1/r-L, then f(d)(i) rapidly decreases with i at any r for all types of inter-ligand interaction. At high ligand concentration (r is close to r(max) = L(-1)), f(d)(0) is close to unity and f(d)(i) rapidly decreases with i for any type of inter-ligand interaction. For strong contact cooperativity, f(d)(0) is close to unity in a much lager r interval ((0.5-1) . r(max)), and f(d)(1) approximately a(o)(-1) at r approximately 0.5 . r(max). In the case of long-range interaction between bound ligands, the dependence f(d)(i) is more complex and has a maximum at i approximately (1/r-L)(1/2) for anti-cooperative binding. f(bc)(i) is maximal at i approximately 1/r-L for all types of binding except the contact cooperativity. A strong asymmetry in the influence of contact cooperativity and anticooperativity on the ligand distribution along DNA is demonstrated.     

Dynamic force measurements of avidin-biotin and streptavdin-biotin interactions using AFM

Using atomic force microscopy (AFM) we performed dynamic force measurements of the adhesive forces in two model systems: avidin-biotin and streptavidin-biotin. In our experiments we used glutaraldehyde for immobilization of (strept)avidin on the tip and biotin on the sample surface. Such interface layers are more rigid than those usually reported in the literature for AFM studies, when (strept)avidin is coupled with biotinylated bovine albumin and biotin with agarose polymers. We determined the dependence of the rupture forces of avidin-biotin and streptavidin-biotin bonds in the range 300-9600 pN/s. The slope of a semilogarithmic plot of this relation changes at about 1700 pN/s. The existence of two different regimes indicates the presence of two activation barriers of these complexes during the dissociation process. The dissociation rates and activation energy barriers, calculated from the Bell model, for the avidin-biotin and streptavidin-biotin interactions are similar to each other for loading rates > 1700 pN/s but they are different from each other for loading rates < 1700 pN/s. In the latter case, the dissociation rates show a higher stability of the avidin-biotin complex than the streptavidin-biotin complex due to a larger outer activation barrier of 0.8 k(B)T. The bond-rupture force is about 20 pN higher for the avidin-biotin pair than for the streptavidin-biotin pair for loading rates < 1700 pN/s. These two experimental observations are in agreement with the known structural differences between the biotin binding pocket of avidin and of streptavidin.     

Chamber properties from transmitral flow: prediction of average and passive left ventricular diastolic stiffness.

A chamber stiffness (K(LV))-transmitral flow (E-wave) deceleration time relation has been invasively validated in dogs with the use of average stiffness [(DeltaP/DeltaV)(avg)]. K(LV) is equivalent to k(E), the (E-wave) stiffness of the parameterized diastolic filling model. Prediction and validation of 1) (DeltaP/DeltaV)(avg) in terms of k(E), 2) early rapid-filling stiffness [(DeltaP/DeltaV)(E)] in terms of k(E), and 3) passive (postdiastasis) chamber stiffness [(DeltaP/DeltaV)(PD)] from A waves in terms of the stiffness parameter for the Doppler A wave (k(A)) have not been achieved. Simultaneous micromanometric left ventricular (LV) pressure (LVP) and transmitral flow from 131 subjects were analyzed. (DeltaP)(avg) and (DeltaV)(avg) utilized the minimum LVP-LV end-diastolic pressure interval. (DeltaP/DeltaV)(E) utilized DeltaP and DeltaV from minimum LVP to E-wave termination. (DeltaP/DeltaV)(PD) utilized atrial systolic DeltaP and DeltaV. E- and A-wave analysis generated k(E) and k(A). For all subjects, noninvasive-invasive relations yielded the following equations: k(E) = 1,401. (DeltaP/DeltaV)(avg) + 59.2 (r = 0.84) and k(E) = 229.0. (DeltaP/DeltaV)(E) + 112 (r = 0.80). For subjects with diastasis (n = 113), k(A) = 1,640. (DeltaP/DeltaV)(PD) - 8.40 (r = 0.89). As predicted, k(A) showed excellent correlation with (DeltaP/DeltaV)(PD); k(E) correlated highly with (DeltaP/DeltaV)(avg). In vivo validation of average, early, and passive chamber stiffness facilitates quantitative, noninvasive diastolic function assessment from transmitral flow.     

Traction force on a kinetochore at metaphase acts as a linear function of kinetochore fiber length

We are investigating the relation between the force pulling a kinetochore poleward and the length of the corresponding kinetochore fiber. It was recognized by Ostergren in 1950 (Hereditas 36:1-19) that the metaphase position of a chromosome could be achieved by a balance of traction forces were proportional to the distance from kinetochore to pole. For the typical chromosome (i.e., a meiotic bivalent or mitotic chromosome) with a single kinetochore fiber extending to each pole, the resultant force (RF) would equal zero when the chromosome lay at the midpoint between the two poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles. For special chromosomes that have unequal numbers of kinetochore fibers extending towards opposite poles, Ostergren’s proposal suggests that RF = 0 when the chromosome is shifted closer to the pole toward which the greater number of kinetochore fibers are pulling. We have measured the force-length relationship in living spindles by analyzing the metaphase positions of experimentally generated multivalent chromosomes having three or four kinetochore fibers. Multivalent chromosomes of varied configurations were generated by γ-irradiation of nymphs of the grasshopper melanoplus differentialis, and their behavior was analyzed in living first meiotic spermocytes. The lengths of kinetochore fibers were determined from time-lapse photographs by measuring the kinetochore-to-pole distances for fully congressed chromosomes just before the onset of anaphase. In our analysis, force (F) along a single kinetochore fiber is expressed by: F = kL(exp), where k is a length-independent proportionality constant, L represents the kinetochore fiber length, and exp is an unknown exponent. The RF on a chromosome is then given by: RF = σk(i)L(i)(exp), where kinetochore fiber lengths in opposite half- spindles are given opposite sign. If forces on a metaphase chromosome are at equilibrium (RF = 0), then for asymmetrical orientations of multivalents we can measure the individual kinetochore fiber lengths (L(i)) and solve for the exponent that yields a resultant force of zero. The value of the exponent relates how the magnitude of force along a kinetochore fiber varies with its length. For six trivalents and one naturally occurring quadrivalent we calculated an average value of exp = 1.06 +/- 0.18. This result is consistent with Ostergren’s hypothesis and indicates that the magnitude of poleward traction force along a kinetochore fiber is directly proportional to the length of the fiber. Our finding suggests that the balance of forces along a kinetochore fiber may be a major factor regulating the extent of kinetochore microtubule assembly.     

Single molecule studies of antibody-antigen interaction strength versus intra-molecular antigen stability

We investigated molecular recognition of antibodies to membrane-antigens and extraction of the antigens out of membranes at the single molecule level. Using dynamic force microscopy imaging and enzyme immunoassay, binding of anti-sendai antibodies to sendai-epitopes genetically fused into bacteriorhodopsin molecules from purple membranes were detected under physiological conditions. The antibody/antigen interaction strength of 70-170 pN at loading rates of 2-50 nN/second yielded a barrier width of x = 0.12 nm and a kinetic off-rate (corresponding to the barrier height) of k(off) = 6s(-1), respectively. Bacteriorhodopsin unfolding revealed a characteristic intra-molecular force pattern, in which wild-type and sendai-bacteriorhodopsin molecules were clearly distinguishable in their length distributions, originating from the additional 13 amino acid residues epitope in sendai purple membranes. The inter-molecular antibody/antigen unbinding force was significantly lower than the force required to mechanically extract the binding epitope-containing helix pair out of the membrane and unfold it (126 pN compared to 204 pN at the same loading rate), meeting the expectation that inter-molecular unbinding forces are weaker than intra-molecular unfolding forces responsible for stabilizing native conformations of proteins.     

Energetics of kayaking at submaximal and maximal speeds

The energy cost of kayaking per unit distance (C(k), kJ x m(-1)) was assessed in eight middle- to high-class athletes (three males and five females; 45-76 kg body mass; 1.50-1.88 m height; 15-32 years of age) at submaximal and maximal speeds. At submaximal speeds, C(k) was measured by dividing the steady-state oxygen consumption (VO(2), l x s(-1)) by the speed (v, m x s(-1)), assuming an energy equivalent of 20.9 kJ x l O(-1)(2). At maximal speeds, C(k) was calculated from the ratio of the total metabolic energy expenditure (E, kJ) to the distance (d, m). E was assumed to be the sum of three terms, as originally proposed by Wilkie (1980): E = AnS + alphaVO(2max) x t-alphaVO(2max) x tau(1-e(-t x tau(-1))), were alpha is the energy equivalent of O(2) (20.9 kJ x l O(2)(-1)), tau is the time constant with which VO(2max) is attained at the onset of exercise at the muscular level, AnS is the amount of energy derived from anaerobic energy utilization, t is the performance time, and VO(2max) is the net maximal VO(2). Individual VO(2max) was obtained from the VO(2) measured during the last minute of the 1000-m or 2000-m maximal run. The average metabolic power output (E, kW) amounted to 141% and 102% of the individual maximal aerobic power (VO(2max)) from the shortest (250 m) to the longest (2000 m) distance, respectively. The average (SD) power provided by oxidative processes increased with the distance covered [from 0.64 (0.14) kW at 250 m to 1.02 (0.31) kW at 2000 m], whereas that provided by anaerobic sources showed the opposite trend. The net C(k) was a continuous power function of the speed over the entire range of velocities from 2.88 to 4.45 m x s(-1): C(k) = 0.02 x v(2.26) (r = 0.937, n = 32).     

In vivo forces generated by finger flexor muscles do not depend on the rate of fingertip loading during an isometric task

Risk factors for activity-related tendon disorders of the hand include applied force, duration, and rate of loading. Understanding the relationship between external loading conditions and internal tendon forces can elucidate their role in injury and rehabilitation. The goal of this investigation is to determine whether the rate of force applied at the fingertip affects in vivo forces in the flexor digitorum profundus (FDP) tendon and the flexor digitorum superficialis (FDS) tendon during an isometric task. Tendon forces, recorded with buckle force transducers, and fingertip forces were simultaneously measured during open carpal tunnel surgery as subjects (N=15) increased their fingertip force from 0 to 15N in 1, 3, and 10s. The rates of 1.5, 5, and 15N/s did not significantly affect FDP or FDS tendon to fingertip force ratios. For the same applied fingertip force, the FDP tendon generated more force than the FDS. The mean FDP to fingertip ratio was 2.4+/-0.7 while the FDS to tip ratio averaged 1.5+/-1.0 (p<0.01). The fine motor control needed to generate isometric force ramps at these specific loading rates probably required similar high activation levels of multiple finger muscles in order to stabilize the finger and control joint torques at the force rates studied. Therefore, for this task, no additional increase in muscle force was observed at higher rates. These findings suggest that for high precision, isometric pinch maneuvers under static finger conditions, tendon forces are independent of loading rate.     

Roles of cell and microvillus deformation and receptor-ligand binding kinetics in cell rolling

Polymorphonuclear leukocyte (PMN) recruitment to sites of inflammation is initiated by selectin-mediated PMN tethering and rolling on activated endothelium under flow. Cell rolling is modulated by bulk cell deformation (mesoscale), microvillus deformability (microscale), and receptor-ligand binding kinetics (nanoscale). Selectin-ligand bonds exhibit a catch-slip bond behavior, and their dissociation is governed not only by the force but also by the force history. Whereas previous theoretical models have studied the significance of these three "length scales" in isolation, how their interplay affects cell rolling has yet to be resolved. We therefore developed a three-dimensional computational model that integrates the aforementioned length scales to delineate their relative contributions to PMN rolling. Our simulations predict that the catch-slip bond behavior and to a lesser extent bulk cell deformation are responsible for the shear threshold phenomenon. Cells bearing deformable rather than rigid microvilli roll slower only at high P-selectin site densities and elevated levels of shear (>or=400 s(-1)). The more compliant cells (membrane stiffness=1.2 dyn/cm) rolled slower than cells with a membrane stiffness of 3.0 dyn/cm at shear rates >50 s(-1). In summary, our model demonstrates that cell rolling over a ligand-coated surface is a highly coordinated process characterized by a complex interplay between forces acting on three distinct length scales.     

Evaluation of UASB reactor performance during start-up operation using synthetic mixed-acid waste

A start-up experiment was performed in a laboratory-scale, upflow anaerobic sludge blanket (UASB) reactor using seed sludge from a domestic waste treatment plant at 3.8-33.3gCODl(-1)day(-1) loading rates. Analysis over the height of the reactor with time showed that the VSS in the reactor was initially differentiated into active and non-active biomass at increasing gas production and upflow velocities, and specific update rates of the volatile fatty acids (VFA) components were pronounced at the bottom 10% of the reactor. During start-up, specific methanogenic activity and chemical oxygen demand (COD) uptake rate increased from 0.075 to 0.75gCOD-CH(4)(gVSS)(-1)day(-1) and from 0.08 to 0.875gCOD removed (gVSS)(-1)day(-1), respectively. When seed sludge from a distillery waste treatment plant was used, improved performance due to a predominance of active biomass was evident when the loading rate was increased from 9.4 to 28.7gCODl(-1)day(-1). The proposed start-up evaluation is an effective tool to successfully monitor performance of UASB reactors.     

Single molecule characterization of P-selectin/ligand binding

P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s(-1). The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s(-1). Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.     

Hydrodynamic effects in fast AFM single-molecule force measurements

Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture individual receptor–ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few m/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 m/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 m/s pulling speed.Abbrevations AFM atomic force micrcoscopy - pN piconewton - BR bacteriorhodopsin - DFS dynamic force spectroscopy - Ig27 immunoglobulin 27 - If27-8 immunoglobulin 27 octameric construct - BFP biomembrane force probe     

Catch bonds govern adhesion through L-selectin at threshold shear

Flow-enhanced cell adhesion is an unexplained phenomenon that might result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a threshold shear to support leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. Low forces decrease L-selectin-PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). We determined that a force-dependent decrease in off-rates dictated flow-enhanced rolling of L-selectin-bearing microspheres or neutrophils on PSGL-1. Catch bonds enabled increasing force to convert short-lived tethers into longer-lived tethers, which decreased rolling velocities and increased the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increased above the optimum, transitions to slip bonds shortened tether lifetimes, which increased rolling velocities and decreased rolling regularity. Thus, force-dependent alterations of bond lifetimes govern L-selectin-dependent cell adhesion below and above the shear optimum. These findings establish the first biological function for catch bonds as a mechanism for flow-enhanced cell adhesion.     

Differences in zero-force and force-driven kinetics of ligand dissociation from beta-galactoside-specific proteins (plant and animal lectins, immunoglobulin G) monitored by plasmon resonance and dynamic single molecule force microscopy

Protein-carbohydrate interactions are involved in diverse regulatory processes. To help understand the mechanics and kinetics of dissociation of receptor-ligand complexes, we have analyzed the separation of lactose and the N-glycan chains of asialofetuin (ASF) from three lectins and an immunoglobulin G fraction by surface plasmon resonance at zero force and by atomic force microscopy with variations of the external force. While the (AB)2 agglutinins from Ricinus communis (RCA) and Viscum album (VAA) show structural homology, the homodimeric galectin-1 from bovine heart (BHL) has no similarity to the two plant lectins except for sharing this monosaccharide specificity. The beta-galactoside-binding immunoglobulin G (IgG) fraction from human serum provides a further model system with distinct binding-site architecture. The k(off) constants for the two plant agglutinins were independent of the nature of the ligand at 1.1-1.3 x 10(-3) s(-1), whereas the geometry of ligand and binding site presentation affected this parameter for BHL (0.5 x 10(-3) s(-1) for lactose and 1 x 10(-3) s(-1) for ASF) and IgG (1.3 x 10(-3) s(-1) for lactose and 0.55 x 10(-3) s(-1) for ASF). When assessing comparatively the rupture forces at a loading rate of 3 nN/s with lactose as ligand, 34 +/- 6 pN (BHL), 36 +/- 4 pN (IgG), 47 +/- 7 pN (VAA), and 58 +/- 9 pN (RCA) were measured. For the same loading rate the rupture forces for the receptor-ASF interactions were found to be 37 +/- 3 pN (BHL), 43 +/- 5 pN (VAA), 45 +/- 6 pN (IgG), and 65 +/- 9 pN (RCA). The variation of the pulling velocity revealed in all cases a linear dependence between the rupture force and the natural logarithm of the loading rate. Performing probability density and Monte Carlo calculations, the potential barrier widths, which determine the inverse dynamic dependence with the rate of force elevation, increased from 4 A (RCA) and 7 A (VAA and IgG) to 10 A (BHL) for the receptor-lactose interactions. Presenting ASF as ligand potential widths of 4 A for RCA and IgG and 6 A for VAA and BHL were obtained. Since the dissociation kinetics at zero force apparently cannot predict the behavior in force-driven experiments, these results reveal new insights into biological functions. The dissociation kinetics under force helps to explain the difference in the toxic potency of VAA and RCA and points to a function of the galectin in cis-crosslinking and in transient trans-bridging.     

Trunk muscle activation and associated lumbar spine joint shear forces under different levels of external forward force applied to the trunk.

High anterior intervertebral shear loads could cause low back injuries and therefore the neuromuscular system may actively counteract these forces. This study investigated whether, under constant moment loading relative to L3L4, an increased externally applied forward force on the trunk results in a shift in muscle activation towards the use of muscles with more backward directed lines of action, thereby reducing the increase in total joint shear force. Twelve participants isometrically resisted forward forces, applied at several locations on the trunk, while moments were held constant relative to L3L4. Surface EMG and lumbar curvature were measured, and an EMG-driven muscle model was used to calculate compression and shear forces at all lumbar intervertebral joints. Larger externally applied forward forces resulted in a flattening of the lumbar lordosis and a slightly more backward directed muscle force. Furthermore, the overall muscle activation increased. At the T12L1 to L3L4 joint, resulting joint shear forces remained small (less than 200N) because the average muscle force pulled backward relative to those joints. However, at the L5S1 joint the average muscle force pulled the trunk forward so that the increase in muscle force with increasing externally applied forward force caused a further rise in shear force (by 102.1N, SD=104.0N), resulting in a joint shear force of 1080.1N (SD=150.4N) at 50Nm moment loading. It is concluded that the response of the neuromuscular system to shear force challenges tends to increase rather than reduce the shear loading at the lumbar joint that is subjected to the highest shear forces.     

Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly

In wild-type soybean LO-1 (WT sLO-1), Asn694 is a weak sixth ligand that is thought to be critical for enzymatic catalysis. In this investigation, N694G sLO-1 was studied to probe its contribution at this sixth ligand position to the kinetic and spectroscopic properties. The k(cat) value of N694G is approximately 230 times lower than that of WT sLO-1 at 25 degrees C, which can be partially explained by a lowered reduction potential of the iron as seen as a shift in the visible ligand-to-metal charge-transfer band (lambda(max) = 410 nm for N694G and lambda(max) = 425 nm for WT sLO-1). This conclusion was supported by a faster rate of oxidation of N694G by the product than that of WT sLO-1 (k(2) = 606 s(-)(1) for N694G and k(2) = 349 s(-)(1) for WT sLO-1). These results suggest a stronger ligand at the active site iron than the native Asn694, which is confirmed to be a water bound to the Fe(II) in the crystal structure. This produces a six-coordinate circular dichroism/magnetic circular dichroism (CD/MCD) spectra for ferrous N694G and an intermediate rhombic electron paramagnetic resonance (EPR) signal for ferric N694G. The EPR spectrum and its pH dependence suggest that the coordination environment of ferric N694G contains one hydroxide and one water. On the basis of both kinetic and structural factors, we propose that the Asn694 water-derived ligand would likely be a hydroxide and the active site, water-derived ligand a water in the ferric state, hence lowering the reaction rate of N694G more than would be expected from the lowered reduction potential alone.     

Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor

Fluorescence correlation spectroscopy (FCS) is suited to determine low concentrations (10(-8) M) of slowly interacting molecules with different translational diffusion coefficients on the level of single molecule counting. This new technique was applied to characterize the interaction dynamics of tetramethylrhodamin labelled alpha-bungarotoxin (B( *)) with the detergent solubilized nicotinic acetylcholine receptor (AChR) of Torpedo californica electric organ. At pseudo-first-order conditions for AChR, the complex formation with B( *) is monophasic. The association rate coefficient of the monoliganded species AChR . B is k(ass)' = 3.8 . 10(3) s(-1) at 293 K (20 degrees C). The dissociation of bound B( *) from the monomer species AChR . B( *) . B (and AChR . B(2)( *)), initiated by adding an excess of nonlabelled alpha-bungarotoxin (B), is biphasic suggesting a three state cascade for the B-sites: R(alpha) --> R(alpha)' --> R(alpha)' with the exchange dissociation constants: (k(diss)')(B) = 5.5(+/-1) . 10(-5) s(-1) and (k(diss)')(B) = 3(+/-1) . 10(-6) s(-1) at 293 K. The data are consistent with dissociative intermediate steps of ligand exchange on two different interconvertible conformations of one binding site. The dissociation of bound B( *) by excess of the neurotransmitter acetylcholine (ACh) is biphasic. At [ACh] = 0.1 M both B( *) are released from the AChR . B(2)( *) species. The mechanism involves associative ternary intermediates (AChR . B( *)A, AChR . B( *)A(2) and AChR . B(2)( *)A(2)). The equilibrium constants (K(A)) and dissociation rate constants (k(-A)) for ACh in the ternary complex state R(alpha)' and R(alpha)', respectively, are K(A)' = 1.1 . 10(-2) M and k(-A)' = 3 . 10(5) s(-1) and K(A)' = 7.5 . 10(-2) M and k(-A)' = 2 . 10(6) s(-1). It is of physiological importance that the FCS data indicate that the AChR monomer species (M(r) = 290 000), which normally at [ACh] 1 mM only binds one ACh molecule, does bind two ACh molecules at [ACh] 0.1 M.     

AM-loading of fluorescent Ca2+ indicators into intact single fibers of frog muscle

The AM loading of a number of different fluorescent Ca2+ indicators was compared in intact single fibers of frog muscle. Among the 13 indicators studied, loading rates (the average increase in the fiber concentration of indicator per first 60 min of loading) varied approximately 100-fold, from approximately 3 microM/h to >300 microM/h (16 degrees C). Loading rates were strongly dependent on the molecular weight of the AM compounds, with the rate increasing steeply as molecular weight decreased below approximately 850. Properties of delta F/F (the Ca2(+)-related fluorescence signal observed with fiber stimulation) were also measured in AM-loaded fibers and compared with those previously reported for fibers microinjected with indicator. In general, the time course of delta F/F was very similar with AM-loading and microinjection; however, the amplitude of delta F/F was usually smaller with AM-loading. There was a strong correlation between the rate of indicator loading and the value of the parameter f (the ratio of the amplitude of delta F/F in AM-loaded versus microinjected fibers). For indicators with small loading rates (<10 microM/h, N = 5), f values were generally small (< or =0.4, N = 4); whereas with large loading rates (>100 microM/h, N = 4), f values were large (> or =0.8, N = 4). This suggests that, with any AM indicator, a small concentration may associate nonspecifically with the fiber (either the indicator is incompletely de-esterified or, if completely de-esterified, not located in the myoplasmic compartment). If the loaded concentration is small, the nonspecific indicator will present a significant source of error in the estimation of [Ca2+]i.     

Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galβ1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen, and the trisaccharide Fucα1,2Galβ1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galβ1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may, thus, play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells.