Urea-induced sequential unfolding of fibronectin: a fluorescence spectroscopy and circular dichroism study

Fibronectin (FN) is an extracellular matrix (ECM) protein found soluble in corporal fluids or as an insoluble fibrillar component incorporated in the ECM. This phenomenon implicates structural changes that expose FN binding sites and activate the protein to promote intermolecular interactions with other FN. We have investigated, using fluorescence and circular dichroism spectroscopy, the unfolding process of human fibronectin induced by urea in different ionic strength conditions. At any ionic strength, the equilibrium unfolding data are well described by a four-state equilibrium model N <= => I(1) <= =>I(2) <= => U. Fitting this model to experimental values, we have determined the free energy change for the different steps. We found that the N <= => I(1) transition corresponds to a free energy of 10.5 +/- 0.4 kcal/mol. Comparable values of free energy change are generally associated with a partial unfolding of the type III domain. For the I(1) <= => I(2) transition, the free energy change is 7.6 +/- 0.4 kcal/mol at low ionic strength but is twice as low at high ionic strength. This result is consistent with observations indicating that the complete unfolding of the type III domain from partially unfolded forms necessitates about 5 kcal/mol. The third step, I(2) <= => U, which leads to the complete unfolding of fibronectin, corresponds to a free energy change of 14.4 +/- 0.9 kcal/mol at low ionic strength whereas this energy is again twice as low under high ionic strength conditions. This hierarchical unfolding of fibronectin, as well as the stability of the different intermediates controlled by ionic strength demonstrated here, could be important for the understanding of activation of the matrix assembly.     

The thermal denaturation of nonpolymerizable alpha alpha-tropomyosin and its segments as a function of ionic strength

Nonpolymerizable tropomyosin (NPTm) is found to unfold thermally at high ionic strength almost exactly as the parent protein, but it does not aggregate at low ionic strength. Thus, NPTm can be used as a tropomyosin surrogate whose coiled-coil structural stability can be probed by varying the ionic strength. Studies of NPTm by CD show that increasing ionic strength stabilizes the coiled-coil structure. CD spectra over a wide range of helix content, obtained by varying either temperature or ionic strength, show an isodichroic point at 203 nm, suggesting a local, residue-level, two-state model. At given temperature, such a local helix in equilibrium random equilibrium suggests ln [phi h/(1-phi h)] = A1 + A2In, wherein phi h is the fraction helix, and A1, A2, and n are constants. In the low ionic strength region, theoretical limiting laws for ionic strength mediated charge-charge, dipole-dipole, and apolar-apolar (salting out) interactions give, respectively, n = 0.5, 1.0, and 1.0. Our experimental values for 40 degrees C, where the data span a wide range of helix content, show n = 1.0, suggesting that ionic strength stabilizes either by reducing dipole-dipole repulsions or by enhancing hydrophobic interactions, both probably interhelix in nature. Two segments of tropomyosin, 11Tm127 and 142Tm281, neither of which aggregate at low ionic strength, give results similar to those for NPTm, i.e., n = 0.96 and 0.84, respectively.     

Reduction kinetics of the ferredoxin-ferredoxin-NADP+ reductase complex: a laser flash photolysis study

The kinetics of reduction of spinach ferredoxin (Fd), ferredoxin-NADP+ reductase (FNR), and the Fd-FNR complex have been investigated by the laser flash photolysis technique. 5-Deazariboflavin semiquinone (5-dRf), generated in situ by laser flash photolysis under anaerobic conditions, rapidly reduced both oxidized Fd (Fdox) (k = 2 X 10(8) M-1 s-1) and oxidized FNR (FNRox) (K = 6.3 X 10(8) M-1 s-1) at low ionic strength (10 mM) at pH 7.0, leading to the formation of reduced Fd (Fdred) and FNR semiquinone (FNR.), respectively. At higher ionic strengths (310 and 460 mM), the rate constant for the reduction of the free Fdox increased about 3-fold (k = 6.7 X 10(8) M-1 s-1 at 310 mM and 6.4 X 10(8) M-1 s-1 at 460 mM). No change in the second-order rate constant for reduction of the free FNRox was observed at high ionic strength. At low ionic strength (10 mM), 5-dRf. reacted only with the FAD center of the preformed 1:1 Fdox-FNRox complex (k = 5.6 X 10(8) M-1 s-1), leading to the formation of FNR.. No direct reduction of Fdox in the complex was observed. No change in the kinetics occurred in the presence of excess NADP+. The second-order rate constant for reduction of Fdox by 5-dRf. in the presence of a stoichiometric amount of fully reduced FNR at low ionic strength was 7 X 10(6) M-1 s-1, i.e., about one-thirtieth the rate constant for reduction of free Fdox.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes

DNA biosensors, especially those based upon detection of the intrinsic negative charge of target DNA, can be greatly improved by the use of uncharged peptide nucleic acid (PNA) probes. Hybridization causes an increased electrostatic barrier for the negatively charged ferri/ferrocyanide redox couple, resulting in an increase in charge transfer resistance R(ct) that is measured using electrochemical impedance spectroscopy. We report on the optimization of PNA probe surface density by the simultaneous co-immobilization of thiol-modified probes and mercaptohexanol, with the PNA surface density controlled by the thiol mole ratio in solution. Maximum R(ct) change upon hybridization is obtained with 10% PNA mole fraction. The effect of the measurement buffer ionic strength is investigated. The electrostatic barrier for charge transfer to the ferri/ferrocyanide redox couple is approximately independent of ionic strength with PNA probes, but greatly increases with decreasing ionic strength, after hybridization with target DNA. This significantly enhances the R(ct) change upon hybridization. The optimization of PNA surface density and measurement buffer ionic strength leads to a 385-fold increase in R(ct) upon hybridization, a factor of 100 larger than previously reported results using either PNA or DNA probes.     

Monitoring the orientation of myosin bridges on two-dimensional maps of birefringence in a single muscle fiber

Two-dimensional maps of birefringence in sarcomers of a single fiber of rabbit m.psoas were obtained by an automated interference microscope developed at our laboratory. The changes in birefringence of muscle fibers reflect the movement of myosin cross-bridges. The orientation of cross-bridges was modified by varying the pH (pH 7.0, 6.0, 8.0) and ionic strength (mu = 0.115, 0.085, 0.235) of the bathing rigor solution. The maximum value of total birefringence in the rigor state was observed at neutral pH. Total birefringence markedly decreased (by 40%) as pH was changed from 7.0 to both 8.0 and 6.0. No significant changes in light phase shifts were found at a 1.5 reduction of ionic strength in the rigor solution. The calculated birefringence values were 45% higher in rigor solutions of a high (mu = 0.235) ionic strength. The results observed are discussed in terms of changes in the orientation of cross-bridges due to the movement of the alpha-helical subfragment-2 away from the filament shaft (pH 8) or coming closer to it (mu = 0.235). The available data do not allow one to explain the results obtained at pH 6.0.     

Kinesin undergoes a 9 S to 6 S conformational transition.

Addition of NaCl or KCl in the presence of 50 nM ATP induces a shift in the sedimentation coefficient (apparent S20,w) of kinesin from 9.4 S at low ionic strength to 6.5 S at high ionic strength. The midpoint for the transition occurs at ionic strength values of 0.39, 0.25, and 0.18 for pH values of 6.3, 6.9, and 8.3, respectively. Gel filtration experiments indicate that the transition to the 6.5 S species is accompanied by a decrease in the diffusion coefficient. Under all conditions which were tested, the 64-kDa beta subunits comigrate with the 120-kDa alpha subunits without any evidence for dissociation of the alpha 2 beta 2 complex. These results are consistent with the change in sedimentation coefficient being due to a conformational transition between a folded form at low ionic strength and an extended form at high ionic strength. This conformational transition is not significantly affected by the nature of the nucleotide bound at the active site since similar results are obtained both in the presence of excess EDTA, which removes the bound ADP, and after replacement of the bound ADP with adenosine 5'-(beta,gamma-imino)triphosphate. The alpha 2 form of kinesin, which lacks the beta subunits, undergoes a similar transition between a 6.7 S form at low ionic strength and a 5.1 S form at high ionic strength with a midpoint for the transition at an ionic strength of 0.5 at pH 6.9. Electron microscopic observation also indicates a transition between a folded conformation at low ionic strength and an extended conformation at high ionic strength for both the alpha 2 beta 2 and alpha 2 species.     

X-ray diffraction testing for weak-binding crossbridges in relaxed bony fish muscle fibres at low ionic strength

Equatorial X-ray diffraction patterns from single skinned fibres from bony fish muscle (turbot) were obtained with the fibres at 6 degrees C bathed in relaxing solutions of 170 down to 26 mM ionic strength. Diffraction patterns from rigor fibres were also obtained as controls. Unlike fibres from rabbit muscle, which show very clear evidence of substantial crossbridge formation at low ionic strength in what is mechanically a rapid equilibrium ("weak-binding") state (Brenner et al., 1982), diffraction patterns from bony fish fibres showed only a small change in relative peak intensities at low ionic strength (26 mM) compared with normal (170 mM) ionic strength. However, there was a slight ordering of the filament lattice at low ionic strength. The specimen temperature used (about 6 degrees C) was not far from the normal physiological temperature of the fish. Likewise, only a small change was seen by Xu et al. (1987) in patterns from frog fibres at low ionic strength at 2 to 6 degrees C. (Rabbit fibres previously studied, where large changes were seen at temperatures of 5 to 20 degrees C, were about 17 to 32 degrees C below physiological.) The I11/I10 ratio for fish fibres at 26 mM ionic strength was actually lower than that for rabbit even at normal ionic strength. This may be associated with an intrinsic structural difference between these muscles or alternatively with the disordering of the crossbridge helix in rabbit muscle found at low temperature by Wray (1987), and could support the view that rabbit fibres at 5 degrees C and normal ionic strength may already have a significant population of weak-binding crossbridges.     

Differential scanning calorimetry of thermal unfolding of the methionine repressor protein (MetJ) from Escherichia coli.

The thermal stability of the methionine repressor protein from Escherichia coli (MetJ) has been examined over a wide range of pH (pH 3.5-10) and ionic strength conditions using differential scanning calorimetry. Under reducing conditions, the transitions are fully reversible, and thermograms are characteristic of the cooperative unfolding of a globular protein with a molecular weight corresponding to the MetJ dimer, indicating that no dissociation of this dimeric protein occurs before unfolding of the polypeptide chains under most conditions. In the absence of reducing agent, repeated scans in the calorimeter show only partial reversibility, though the thermodynamic parameters derived from the first scans are comparable to those obtained under fully reversible conditions. The protein is maximally stable (Tm 58.5 degrees C) at about pH 6, close to the estimated isoelectric point, and stability is enhanced by increasing ionic strength in the range I = 0.01-0.4 M. The average calorimetric transition enthalpy (delta Hm) for the dimer is 505 +/- 28 kJ mol-1 under physiological conditions (pH 7, I = 0.125, Tm = 53.2 degrees C) and shows a small temperature dependence which is consistent with an apparent denaturational heat capacity change (delta Cp) of about +8.9 kJ K-1 mol-1. The effects of both pH and ionic strength on the transition temperature and free energy of MetJ unfolding are inconsistent with any single amino acid contribution and are more likely the result of more general electrostatic interactions, possibly including significant contributions from electrostatic repulsion between the like-charged monomers which can be modeled by a Debye-Hückel screened potential.     

Kinetics properties of Cu,Zn-superoxide dismutase as a function of metal content

The kinetics of bovine Cu,Zn superoxide dismutase were studied by pulse radiolysis. To ensure the absence of catalytically active free copper, commercially obtained holo-superoxide dismutase was demetallated, and the apo-superoxide dismutase concentrations were determined by isothermal titration calorimetry prior to reconstitution with defined amounts of copper and zinc. The catalytic rate constant was determined as a function of ionic strength over the range of 4-154 mM, and of the copper and zinc content. The catalytic rate constant increases with ionic strength up to (1.5 +/- 0.2) x 10(9) M(-1) s(-1) at an ionic strength of 15 mM, and then decreases. At pH 7 and 50 mM ionic strength, k = (1.2 +/- 0.2) x 10(9) M(-1) s(-1), and at a physiologically relevant ionic strength of 150 mM, it is (0.7 +/- 0.1) x 10 (9) M(-1) s(-1). The effect of ionic strength is ascribed to the inhomogeneous electric field generated by the surface charges of superoxide dismutase. The value of the catalytic rate constant at 50 mM is ca. 2-fold smaller than earlier values reported in the literature. The relationship between copper content and the catalytic rate constant shows that addition of more than a stoichiometric amount of copper cannot be masked efficiently by EDTA. The possibility exists that earlier reported values were based on experiments contaminated with trace amounts of copper.     

Effect of ionic strength on the transfer of 1-pyrenemethyl-3 beta-hydroxy-22,23-bisnor-5-cholenate between bilayer vesicles containing phosphatidylserine.

The influence of ionic strength or the concentration of K+ ([K+]) of the aqueous phase on the spontaneous transfer of cholesterol between negatively charged bilayer vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) (1:1, mole:mole) was studied using a pyrene-labelled cholesterol analogue, 1-pyrenemethyl-3 beta-hydroxy-22,23-bisnor-5-cholenate (PMC), as the probe. The decrease in PMC excimer fluorescence was best fitted to a bi-exponential function. Increasing [K+] from 0.1 M to 0.3 M had little effect on the shorter half-time (1.4 +/- 0.2 min) but increased the longer half-time from 16.3 +/- 1.9 min to 26.7 +/- 2.1 min. Fluorescence quenching and titration of an interface-located fluorophore, 1-anilinonaphthalene-8-sulfonic acid (ANS) revealed an increase in interfacial hydrophobicity upon increasing in ionic strength. The physical state of the acyl chains was not affected by ionic strength as indicated by a constant PMC excimer:monomer fluorescence intensity ratio. However, an increase in enthalpy change of the lipid phase transition from 15.7 kJ/mol ([K+] = 0.1 M) to 21.3 kJ/mol ([K+] = 0.3 M), together with a slight increase in the transition temperature, implies that interactions between adjacent molecules in the charged lipid bilayer vesicles became stronger at higher ionic strength. Our results suggest that the van der Waals attraction between PMC and phospholipid molecules could be affected by conformation changes in the charged head group region brought about by changes of ionic strength in the aqueous phase, with consequent effects on the desorption of cholesterol from the bilayer surface.     

Effect of ionic strength and pH on the activity of pyruvate dehydrogenase complex from pig kidney cortex.

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex is sensitive to changes in ionic strength (mu). At low ionic strength (mu = 0.04 M) the specific activity of PDC was 12.22 mumol/min/mg, whereas at high ionic strength (mu = 0.15 M) the measured activity of the complex decreased to 4.88 mumol/min/mg. The optimum activity of PDC was achieved within a small range of ionic strength, mu = 0.035-0.040 M. Increasing the ionic strength from mu = 0.05 to mu = 0.15 M decreased the s0.5 for pyruvate from 125 to 72 microM and increased the Hill coefficient from 1.0 to 1.3. The effect of pH on PDC activity also was dependent upon ionic strength. At pH 7.2 the activity of PDC at mu = 0.05 and mu = 0.15 M was 90 and 55% of the maximal activity, respectively. Furthermore, the effects of Na+, K+, HCO3-, Cl-, and HPO4(2-) on PDC activity were dependent on ionic strength and pH. The addition of K+ (80 mM) at mu = 0.10 and mu = 0.15 M increased the activity of PDC by 12 and 42%, respectively. Lowering the pH from 8.2 to 7.5 resulted in a decrease in the s0.5 for pyruvate from 179 to 110 microM and from 110 to 35 microM in the presence and absence of K+ (80 mM), Na+ (20 mM), Cl- (20 mM), HCO3- (20 mM), and HPO4(2-) (10 mM), respectively. The observed changes in the properties of PDC in response to changes in ionic strength likely was a result of changes in the intramolecular electrostatic interactions within the complex. In this regard it was determined using two-dimensional agarose gel electrophoresis of the intact multienzyme complex that increasing the ionic strength to which PDC is exposed decreased the measured radius of PDC and may have decreased the electronegative surface charge of the complex.     

Effect of ionic strength on the organization and dynamics of tryptophan residues in erythroid spectrin: a fluorescence approach

The ionic strength of the medium plays an important role in the structure and conformation of erythroid spectrin. The spectrin dimer is a flexible rod at physiological ionic strength. However, lower ionic strength results in elongation and rigidification (stiffening) of spectrin as shown earlier by electron microscopy and hydrodynamic studies. The ionic strength induced structural transition does not involve any specific secondary structural changes. In this article, we have used a combination of fluorescence spectroscopic approaches that include red edge excitation shift (REES), fluorescence quenching, time-resolved fluorescence measurements, and chemical modification of the spectrin tryptophans to assess the environment and dynamics of tryptophan residues of spectrin under different ionic strength conditions. Our results show that while REES, fluorescence anisotropy, lifetime, and chemical modification of spectrin tryptophans remain unaltered in low and high ionic strength conditions, quenching of tryptophan fluorescence by the aqueous quencher acrylamide (but not the hydrophobic quencher trichloroethanol) and resonance energy transfer to a dansyl-labeled fatty acid show differences in tryptophan environment. These results, which report tertiary structural changes in spectrin upon change in ionic strength, are relevant in understanding the molecular details underlying the conformational flexibility of spectrin.     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increased or decreased to alter the electrostatic interaction between enzyme and substrate in the lytic action of human lysozyme using site-directed mutagenesis. The amino acid substitutions accompanying either the addition or the removal of two units of positive charge have shifted the optimal ionic strength (NaCl concentration in 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticus cell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively. In addition to the change in ionic strength-activity profile, the pH-activity profile and the effect of a polycationic electrolyte, poly-L-Lys-HCl, on the lytic activity were significantly changed. Owing to the shifts in both ionic strength profiles and pH profiles the Arg74/Arg126 mutant has become a better catalyst than wild-type enzyme under the conditions of high ionic strength and high pH, and the Gln41/Ser101 mutant has become a better catalyst under the conditions of low ionic strength and low pH.     

Ionic strength and pH dependence of the reaction between plastocyanin and Photosystem 1. Evidence of a rate-limiting conformational change

The electron-transfer reaction between spinach wild-type plastocyanin (Pc(WT)) two site-directed mutants, Pc(Thr79His) and Pc(Lys81His), and spinach Photosystem 1 particles, has been studied as a function of protein concentration, ionic strength and pH by using laser-flash absorption spectroscopy. The kinetic data are interpreted using the simplest possible three-step model, involving a rate-limiting conformational change preceding intracomplex electron transfer. The three proteins show similar concentration, pH and ionic strength dependencies. The effects of ionic strength and pH on the reaction indicate a strong influence of complementary charges on complex formation and stabilization. Studies with apoprotein support the opinion that the hydrophobic patch is critical for an productive interaction with the reaction center of Photosystem 1. Together with earlier site-directed mutagenesis studies, the absence of a detectable Photosystem 1 reaction in the presence of reduced azurin, stellacyanin, cytochrome c and cytochrome c₅₅₁, demonstrates the existence of a high level of specificity in the protein-protein interface in the formation of an efficient electron-transfer complex.     

Protein dissociation from DNA in model systems and chromatin.

Salt induced dissociation of protamine, poly(L-lysine) and poly(L-arginine) from DNA was measured by relative light scattering at theta = 90 degrees and/or centrifugation. Dissociation of histones from DNA was studied using relative light scattering and intrinsic tyrosine fluorescence. Protamine was dissociated from DNA at 0.15 M MgCl2 (ionic strength mu = 0.45) or 0.53 M NaCl (mu = 0.53) based on light scattering data and at approximately 0.2 M MgCl2 (mu = 0.6) or 0.6 M NaCl based on centrifugation data. NaCl induced dissociation of poly(Lys) or poly(Arg) from natural DNAs measured by light scattering did not depend on the guanine plus cytosine content. To dissociate poly(Arg) from DNA higher ionic strength using NaCl, MgCl2, or CaCl2, similar ionic strength using NaClo4, and lower ionic strength using Na2SO4 was needed then to dissociated poly(Lys). Both the decrease in light scattering and the enhancement of tyrosine fluorescence of chromatin occurred between 0.5 and 1.5 M NaCl when histones were dissociated.     

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.     

Effect of ionic strength and oxidation on the P-loop conformation of the protein tyrosine phosphatase-like phytase, PhyAsr

The protein tyrosine phosphatase (PTP)-like phytase, PhyAsr, from Selenomonas ruminantium is a novel member of the PTP superfamily, and the only described member that hydrolyzes myo-inositol-1,2,3,4,5,6-hexakisphosphate. In addition to the unique substrate specificity of PhyAsr, the phosphate-binding loop (P-loop) has been reported to undergo a conformational change from an open (inactive) to a closed (active) conformation upon ligand binding at low ionic strength. At high ionic strengths, the P-loop was observed in the closed, active conformation in both the presence and absence of ligand. To test whether the P-loop movement can be induced by changes in ionic strength, we examined the effect that ionic strength has on the catalytic efficiency of PhyAsr, and determined the structure of the enzyme at several ionic strengths. The catalytic efficiency of PhyAsr is highly sensitive to ionic strength, with a seven-fold increase in k(cat)/K(m) and a ninefold decrease in K(m) when the ionic strength is increased from 100 to 500 mm. Surprisingly, the P-loop is observed in the catalytically competent conformation at all ionic strengths, despite the absence of a ligand. Here we provide structural evidence that the ionic strength dependence of PhyAsr and the conformational change in the P-loop are not linked. Furthermore, we demonstrate that the previously reported P-loop conformational change is a result of irreversible oxidation of the active site thiolate. Finally, we rationalize the observed P-loop conformational changes observed in all oxidized PTP structures.     

Study of electron transport in heme proteins. X. Effect of pH, ionic strength, and zinc ions and the rate of ferricytochrome c reduction by oxymyoglobin from swine heart]

The rate of the redox reaction between porcine MbO2 and ferri-Cyt c at different ionic strengths in the pH range 5-8 has been studied. At low ionic strength (I = 0-0.1) the pH dependence curve was found to have a sigmoid shape with pKeff approximately 5.7, implying the effect of ionization of His-119(GH1) at the "active site" of myoglobin on the kinetics of the process. In this range of ionic strengths the rate of the reaction decreases sharply. The slope of the curve in the coordinates of IgKexp versus square root of I/1 + square root of I varies depending on pH. It is greater at pH less than or equal to 6 and smaller at pH 7.5, which is due to deprotonation of His(GH1). At high ionic strength (I greater than 0.1) the rate of electron transfer is negligible, independent of pH and does not practically change as I increases from 0.1 to 1. It is shown that the local electrostatic interactions play a decisive role in the formation of an efficient electron-transfer complex between Mb and Cyt c. The binding of the zinc ion to His(GH1) was found to inhibit the electron transfer at I = 0.01, similarly to what occurs at a high ionic strength, though the "reactive" charges of the proteins are not screened and the positive charge at His(GH1) is retained. This suggests that His(GH1) is directly involved in the mechanism of electron transfer from Mb to Cyt c. The data obtained are compared with earlier data on the effect of pH, ionic strength and zinc ions on the reaction between MbO2 from sperm whale and Cyt c. To explain the higher efficiency of pig MbO2 as electron donor, the electrostatic and steric properties of both myoglobins have been analyzed.     

Interaction of proteins in solution from small-angle scattering: a perturbative approach

In this work an improved methodology for studying interactions of proteins in solution by small-angle scattering is presented. Unlike the most common approach, where the protein-protein correlation functions g(ij)(r) are approximated by their zero-density limit (i.e., the Boltzmann factor), we propose a more accurate representation of g(ij)(r) that takes into account terms up to the first order in the density expansion of the mean-force potential. This improvement is expected to be particularly effective in the case of strong protein-protein interactions at intermediate concentrations. The method is applied to analyze small-angle x-ray scattering data obtained as a function of the ionic strength (from 7 to 507 mM) from acidic solutions of beta-lactoglobulin at the fixed concentration of 10 gl(-1). The results are compared with those obtained using the zero-density approximation and show significant improvement, particularly in the more demanding case of low ionic strength.     

Ionic-strength dependence of the conformational change in the unphosphorylated sodium pump

The conformational change in the unphosphorylated sodium pump was studied as a function of ionic strength to learn whether the rate of the reaction is affected. The results corroborate our proposal [Smirnova, I. N., Lin, S.-H., and Faller, L. D. (1995) Biochemistry 34, 8657-8667] that competitive binding of the transported ions to two (or more) equivalent sites regulates a concerted change in protein conformation. An approximately 10-fold increase in ionic strength decreased the intrinsic affinity of the Na+ conformation of the enzyme for both Na+ and K+ roughly 3-fold, decreased the rate of the change from Na+ to K+ conformation by more than half, and increased the rate of the reverse reaction by about an order of magnitude. The logarithm of the first-order rate constant for the change from Na+ to K+ conformation depended inversely upon the square root of the ionic strength with the extrapolated value at zero ionic strength expressed as a second-order rate constant (1.1 x 10(9) M(-1) sec(-1)) approaching the limit for a diffusion-controlled reaction. The first-order rate constant for the change from K+ to Na+ conformation depended directly upon ionic strength and extrapolated to a zero-ionic-strength value (0.002 s(-1)) far below the diffusion limit. The results are compatible with shielding of oppositely charged domains that move through the solvent when the pump cycles between conformations. Electrostatic interactions between domains evidently contribute to the driving force for the change from Na+ to K+ conformation and to the stability of the K+ conformation.