Adhesion of mussel foot protein mefp-5 to mica: an underwater superglue

Mussels have a remarkable ability to attach their holdfast, or byssus, opportunistically to a variety of substrata that are wet, saline, corroded, and/or fouled by biofilms. Mytilus edulis foot protein-5 (Mefp-5) is one of several proteins in the byssal adhesive plaque of the mussel M. edulis. The high content of 3,4-dihydroxyphenylalanine (Dopa) (～30 mol %) and its localization near the plaque-substrate interface have often prompted speculation that Mefp-5 plays a key role in adhesion. Using the surface forces apparatus, we show that on mica surfaces Mefp-5 achieves an adhesion energy approaching E(ad) = ～-14 mJ/m(2). This exceeds the adhesion energy of another interfacial protein, Mefp-3, by a factor of 4-5 and is greater than the adhesion between highly oriented monolayers of biotin and streptavidin. The adhesion to mica is notable for its dependence on Dopa, which is most stable under reducing conditions and acidic pH. Mefp-5 also exhibits strong protein-protein interactions with itself as well as with Mefp-3 from M. edulis.     

Ionic currents and ion fluxes in Neurospora crassa hyphae

Voltage dependence of ionic currents and ion fluxes in a walled, turgor-regulating cell were measured in Neurospora crassa. The hyphal morphology of the model organism Neurospora simplifies cable analysis of ionic currents to determine current density for quantitative comparisons with ion fluxes. The ion fluxes were measured directly and non-invasively with self-referencing ion-selective microelectrodes. Four ions (H(+), Ca(2+), K(+), and Cl(-)) were examined. H(+) net uptake and Ca(2+) net release were small (10.2 nmol m(-2) s(-1) and 1.1 nmol m(-2) s(-1), respectively) and voltage independent. K(+) and Cl(-) fluxes were larger and voltage dependent. Maximal K(+) net release ( approximately 1440 nmol m(-2) s(-1)) was observed at positive voltages (+15 mV), while maximal Cl(-) net release ( approximately 905 nmol m(-2) s(-1)) was observed at negative voltage (-210 mV). A possible function of the net outward K(+) and Cl(-) fluxes is regulation of the plasma membrane potential. Total ion fluxes were 37-58% of the total ionic current density (about +/-244 mA m(-2), equivalent to +/-2500 nmol m(-2) s(-1), at 0 mV and -200 mV) so other ions must contribute significantly to the ionic currents.     

Salt sensitivity in wheat : a case for specific ion toxicity

Two selected lines of bread wheat, Triticum aestivum L., differing in their relative salt resistance, were grown in isosmotic solutions of different ionic compositions to investigate sensitivity to specific ions. Growth rates and ion accumulation were determined. The salt composition of the various solutions had little effect on the growth of the salt-resistant line, but significantly affected that of the salt-sensitive line. Specifically, solutions containing high Na⁺ concentrations were more toxic than those containing high Cl⁻ concentrations or high concentrations of nutrient ions. There were few differences in ion accumulation between lines in a given treatment, although the sensitive line tended to accumulate more Na⁺ than the tolerant line in the salt treatments with high Na⁺ concentrations. The overall results provide evidence that there is a definite specific ion effect which is related to salt sensitivity in wheat. It is suggested that superior compartmentation of toxic ions, principally Na⁺, may be a mechanism of salt resistance in this case.     

The influence of cross-linking on protein-protein interactions in a marine adhesive: the case of two byssus plaque proteins from the blue mussel

The interaction between two proteins, Mefp-1 and Mefp-2, from the byssal plaque of the blue mussel, Mytilus edulis, was investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The challenge in using a surface-sensitive technique to investigate the interaction between two strongly adhesive proteins was met by coupling a biotinylated version of one of the proteins (b-Mefp-1) to an inert two-dimensional arrangement of streptavidin (SA) formed on top of a biotin-doped supported phospholipid bilayer. The interaction between Mefp-1 and Mefp-2 was further investigated by addition of Mefp-2 to SA-coupled b-Mefp-1, where the latter was either in the native state or cross-linked using sodium periodate (NaIO(4)), Cu(2+), or mushroom tyrosinase. With this coupling strategy it is shown that a requirement for attraction between the two proteins is that tyrosinase is used as the cross-linking agent of b-Mefp-1. By inhibiting the enzymatic activity of tyrosinase it is also shown that enzymatic activity is required for both efficient binding of tyrosinase to SA-coupled b-Mefp-1 as well as for the subsequent binding of Mefp-2. In contrast, spontaneous adsorption of Mefp-1 to a methyl-terminated (thiolated) gold surface followed by addition of Mefp-2 results in binding of Mefp-2 for all cross-linking agents. This suggests that cross-linking of Mefp-1 adsorbed on a solid surface induces structural changes in the adsorbed protein layer, resulting in exposure of free surface patches on which Mefp-2 binds.     

Effect of Ionic Strength on the Kinetics of Trypsin and Alpha Chymotrypsin

The kinetic effects resulting from changes in the medium ionic strength on reactions involving trypsin or α-chymotrypsin are different. The reaction rate increases continuously as the ionic strength increases with α-chymotrypsin. With trypsin, the rate increases at low ionic strengths but as the ionic strength further increases a gradual inhibitory effect is observed. The effects produced by different salts of various valence types (from uni-univalent to uni-trivalent or tri-univalent) are essentially the same, and they are a function of the square root of the ionic strength. The quantitative differences among the various salts may be accounted for on the basis of individual properties of the ions, such as the size of the hydrated ion, "association," etc. The effects of salts on the enzymic reactions described herein are amenable to the same electrostatic treatment applicable to non-enzymatic reactions. By applying Brönsted's basic kinetic concepts and the Debye-Hückel law of electrolyte activity, it appears that the salt effects are mainly due to changes in the dissociation of ionizable groups. This appears to be a general method for analyzing the effect of inorganic ions on enzymic reactions.     

A model study of factors involved in adhesion of Pseudomonas fluorescens to meat.

A study was undertaken to investigate the factors involved in the adhesion of Pseudomonas fluorescens to model meat surfaces (tendon slices). Adhesion was fast (less than 2.5 min) and was not suppressed by killing the cells with UV, gamma rays, or heat, indicating that physiological activity was not required. In various salt solutions (NaCl, KCl, CaCl2, MgCl2), adhesion increased with increasing ionic strength up to 10 to 100 mM, suggesting that, at low ionic strengths, electrostatic interactions were involved in the adhesion process. At higher ionic strengths (greater than 10 to 100 mM) or in the presence of Al3+ ions, adhesion was sharply reduced. Selectively blocking of carboxyl or amino groups at the cell surface by chemical means did not affect adhesion. These groups are therefore not directly involved in an adhesive bond with tendon. Given a sufficient cell concentration (10(10) CFU.ml-1) in the adhesion medium, the surface of tendon was almost entirely covered with adherent bacteria. This suggests that if the adhesion is specific, the attachment sites on the tendon surface must be located within collagen or proteoglycan molecules.     

A model study of factors involved in adhesion of Pseudomonas fluorescens to meat.

A study was undertaken to investigate the factors involved in the adhesion of Pseudomonas fluorescens to model meat surfaces (tendon slices). Adhesion was fast (less than 2.5 min) and was not suppressed by killing the cells with UV, gamma rays, or heat, indicating that physiological activity was not required. In various salt solutions (NaCl, KCl, CaCl2, MgCl2), adhesion increased with increasing ionic strength up to 10 to 100 mM, suggesting that, at low ionic strengths, electrostatic interactions were involved in the adhesion process. At higher ionic strengths (greater than 10 to 100 mM) or in the presence of Al3+ ions, adhesion was sharply reduced. Selectively blocking of carboxyl or amino groups at the cell surface by chemical means did not affect adhesion. These groups are therefore not directly involved in an adhesive bond with tendon. Given a sufficient cell concentration (10(10) CFU.ml-1) in the adhesion medium, the surface of tendon was almost entirely covered with adherent bacteria. This suggests that if the adhesion is specific, the attachment sites on the tendon surface must be located within collagen or proteoglycan molecules.     

Role of a conserved salt bridge between the PAS core and the N-terminal domain in the activation of the photoreceptor photoactive yellow protein

The effect of ionic strength on the conformational equilibrium between the I(2) intermediate and the signaling state I(2)' of the photoreceptor PYP and on the rate of recovery to the dark state were investigated by time-resolved absorption and fluorescence spectroscopy. With increasing salt concentration up to approximately 600 mM, the recovery rate k(3) decreases and the I(2)/I(2)' equilibrium (K) shifts in the direction of I(2)'. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl(2), MgSO(4)) salts show that the low salt effect depends on the ionic strength and not on the cation or anion species. These observations can be described over the entire ionic strength range by considering the activity coefficients of an interdomain salt bridge. At low ionic strength the activity coefficient decreases due to counterion screening whereas at high ionic strength binding of water by the salt leads to an increase in the activity coefficient. From the initial slopes of the plots of log k(3) and log K versus the square root of the ionic strength, the product of the charges of the interacting groups was found to be -1.3 +/- 0.2, suggesting a monovalent ion pair. The conserved salt bridge K110/E12 connecting the beta-sheet of the PAS core and the N-terminal domain is a prime candidate for this ion pair. To test this hypothesis, the mutants K110A and E12A were prepared. In K110A the salt dependence of the I(2)/I(2)' equilibrium was eliminated and of the recovery rate was greatly reduced below approximately 600 mM. Moreover, at low salt the recovery rate was six times slower than in wild-type. In E12A significant salt dependence remained, which is attributed to the formation of a novel salt bridge between K110 and E9. At high salt reversal occurs in both mutants suggesting that salting out stabilizes the more compact I(2) structure. However, chaotropic anions like SCN shift the I(2)/I(2)' equilibrium toward the partially unfolded I(2)' form. The salt linkage K110/E12 stabilizes the photoreceptor in the inactive state in the dark and is broken in the light-induced formation of the signaling state, allowing the N-terminal domain to detach from the beta-scaffold PAS core.     

Ionic volumes in solution

The volumetric properties of electrolytes in solutions indicate the interactions of the constituent ions with their environment: the solvent and other ions. The interactions with the solvent alone are manifested at infinite dilution by the standard partial molar volume, V(infinity)(salt), obtained from density measurements. To study the interactions, it is necessary to split V(infinity)(salt) into the additive ionic contributions, V(infinity)(ion), using an extra-thermodynamic assumption. Values of V(infinity)(ion) for small ions depend cardinally on the electrostriction of the solvent that can be obtained from an iterative shell-by-shell calculation from a continuum model of the solvent. The solvent shrinkage per mol of ions is DeltaV(el)(ion)<0. Also, the molar electrostriction of the solvent S, DeltaV(el)(S)<0, is calculable. The ratio DeltaV(el)(ion)/DeltaV(el)(S)=n(infinity) is the solvation number of the ion in S at infinite dilution. The calculated V(infinity)(ion)(calc) are compared with the experimental values, showing good agreement for many univalent ions in both single solvents and in some binary solvent mixtures, where no appreciable preferential solvation takes place. Ion pairing sets in under certain circumstances of ionic charge and solvent permittivity. The difference DeltaV(ip)=V(ip)(infinity)-[V(infinity)(+)+V(infinity)(-)]>0 is obtained experimentally from the pressure derivative of the association constant. The ratio Deltan(ip)=DeltaV(ip)/DeltaV(el)(S) represents the number of solvent molecules released to the bulk on ion pairing by the diminution of the electrostriction.     

Electrical potentials of plant cell walls in response to the ionic environment

Electrical potentials in cell walls (psi(Wall)) and at plasma membrane surfaces (psi(PM)) are determinants of ion activities in these phases. The psi(PM) plays a demonstrated role in ion uptake and intoxication, but a comprehensive electrostatic theory of plant-ion interactions will require further understanding of psi(Wall). psi(Wall) from potato (Solanum tuberosum) tubers and wheat (Triticum aestivum) roots was monitored in response to ionic changes by placing glass microelectrodes against cell surfaces. Cations reduced the negativity of psi(Wall) with effectiveness in the order Al(3+) > La(3+) > H(+) > Cu(2+) > Ni(2+) > Ca(2+) > Co(2+) > Cd(2+) > Mg(2+) > Zn(2+) > hexamethonium(2+) > Rb(+) > K(+) > Cs(+) > Na(+). This order resembles substantially the order of plant-root intoxicating effectiveness and indicates a role for both ion charge and size. Our measurements were combined with the few published measurements of psi(Wall), and all were considered in terms of a model composed of Donnan theory and ion binding. Measured and model-computed values for psi(Wall) were in close agreement, usually, and we consider psi(Wall) to be at least proportional to the actual Donnan potentials. psi(Wall) and psi(PM) display similar trends in their responses to ionic solutes, but ions appear to bind more strongly to plasma membrane sites than to readily accessible cell wall sites. psi(Wall) is involved in swelling and extension capabilities of the cell wall lattice and thus may play a role in pectin bonding, texture, and intercellular adhesion.     

Surface-enhanced Raman spectroscopy of DOPA-containing peptides related to adhesive protein of marine mussel, Mytilus edulis

The common blue marine mussel adheres to underwater surfaces using an adhesive protein (Mefp-1) extruded from its foot. This highly hydroxylated protein contains a number of unusual amino acids, including 3,4-dihydroxyphenylalanine (DOPA), which is thought to contribute to the crosslinking of the extruded threads and adhesion to the substratum. Mefp-1 adheres to a wide variety of surfaces and is ultimately biodegradable. In this study we use surface-enhanced Raman spectroscopy (SERS) to characterize the adsorption of DOPA-containing peptides on colloidal gold. The peptides are simplified fragments of the Mefp-1 consensus decapeptide repeat, Ala-Lys-Pro-Ser-Tyr-DHP-Hyp-Thr-DOPA-Lys. Our results show that the peptides TDeltaKA, PTDeltaKA, and PPTDeltaKA (where Delta represents DOPA) coordinate to the gold surface through the catechol oxygens of the DOPA residue and through primary amine groups. The diproline sequence introduces conformational constraints that influence the conformations of the adsorbed peptides. These findings lay the groundwork for developing synthetic adhesives for underwater and medical applications.     

Utility of anion and cation combinations for phasing of protein structures

We report the use of anionic (I(-)), cationic (Ba(2+), Cd(2+)) and ionic mixtures (I(-) plus Ba(2+)) for derivatizing liver fatty acid binding protein (LFABP) crystals. Use of cationic and anionic salts in phasing experiments revealed distinct non-overlapping sites for these ions, suggesting exclusive binding regions on LFABP. Interestingly, cations of identical charge and valency (like Ba(2+) and Cd(2+)) bound to distinct pockets on the protein surface. Furthermore, a mixture of salts containing both I(-) and Ba(2+) was very useful in phasing experiments as these oppositely charged ions bound to different regions of LFABP. Our data therefore suggest that cationic and anionic salt mixtures like BaCl(2) with NH(4)I or salts like CdI, BaI where each ion has a significant anomalous signal for a given X-ray wavelength may be valuable reagents for phasing during structure determination.     

Stability of Secondary and Tertiary Structures of Virus-Like Particles Representing Noroviruses: Effects of pH,Ionic Strength,and Temperature and Implications for Adhesion to Surfaces

Loss of ordered molecular structure in proteins is known to increase their adhesion to surfaces. The aim of this work was to study the stability of norovirus secondary and tertiary structures and its implications for viral adhesion to fresh foods and agrifood surfaces. The pH, ionic strength, and temperature conditions studied correspond to those prevalent in the principal vehicles of viral transmission (vomit and feces) and in the food processing and handling environment (pasteurization and refrigeration). The structures of virus-like particles representing GI.1, GII.4, and feline calicivirus (FCV) were studied using circular dichroism and intrinsic UV fluorescence. The particles were remarkably stable under most of the conditions. However, heating to 65°C caused losses of β-strand structure, notably in GI.1 and FCV, while at 75°C the α-helix content of GII.4 and FCV decreased and tertiary structures unfolded in all three cases. Combining temperature with pH or ionic strength caused variable losses of structure depending on the particle type. Regardless of pH, heating to pasteurization temperatures or higher would be required to increase GII.4 and FCV adhesion, while either low or high temperatures would favor GI.1 adhesion. Regardless of temperature, increased ionic strength would increase GII.4 adhesion but would decrease GI.1 adhesion. FCV adsorption would be greater at refrigeration, pasteurization, or high temperature combined with a low salt concentration or at a higher NaCl concentration regardless of temperature. Norovirus adhesion mediated by hydrophobic interaction may depend on hydrophobic residues normally exposed on the capsid surface at pH 3, pH 8, physiological ionic strength, and low temperature, while at pasteurization temperatures it may rely more on buried hydrophobic residues exposed upon structural rearrangement.     

Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp recording

Patch-clamp recording has revolutionized the study of ion channels, transporters, and the electrical activity of small cells. Vital to this method is formation of a tight seal between glass recording pipette and cell membrane. To better understand seal formation and improve practical application of this technique, we examine the effects of divalent ions, protons, ionic strength, and membrane proteins on adhesion of membrane to glass and on seal resistance using both patch-clamp recording and atomic force microscopy. We find that H(+), Ca(2+), and Mg(2+) increase adhesion force between glass and membrane (lipid and cellular), decrease the time required to form a tight seal, and increase seal resistance. In the absence of H(+) (10(-10) M) and divalent cations (<10(-8) M), adhesion forces are greatly reduced and tight seals are not formed. H(+) (10(-7) M) promotes seal formation in the absence of divalent cations. A positive correlation between adhesion force and seal formation indicates that high resistance seals are associated with increased adhesion between membrane and glass. A similar ionic dependence of the adhesion of lipid membranes and cell membranes to glass indicates that lipid membranes without proteins are sufficient for the action of ions on adhesion.     

Differential scanning calorimetric studies on bovine serum albumin: II. Effects of neutral salts and urea

Using defatted and SH-blocked bovine serum albumin (BSA), measurements of differential scanning calorimetry (d.s.c.) have been made mainly in NaSCN solution. BSA undergoes a heat-induced conformational transition in a particular range of pH and ionic strength and is separated into two thermally independent units, each of which has different thermostability in acidic and alkaline pH regions. Comparisons were made of the pH dependencies of the enthalpy of thermal denaturation (ΔH) and the temperature of thermal denaturation (T_d) in 0.01 NaSCN with those in 0.01 NaCl. It has been found that the stabilizing effect of NaSCN on BSA is larger than that of NaCl at pH 3.5–8, and that the heat-induced transition occurs by the electrostatic repulsive forces among the positively charged amino acid residues in a segment Arg 184–Arg 216 containing Trp 212 and the primary binding sites of anions. At ionic strength 0.01, the relative effectiveness of anions in suppressing the heat-induced transition and increasing the thermostability of BSA follows the order ClO₄⁻ − SCN⁻ > I⁻ > SO₄²⁻ > Br⁻ > Cl⁻. At ionic strength 0.1, the heat-induced transition is suppressed in all the salt solutions, and a T_d increase follows the order ClO₄⁻ SCN⁻ > I⁻ > Br⁻ > Cl⁻ SO₄²⁻. Thus, the highly chaotropic ions thermostabilize BSA more markedly than kosmotropic ions in the low and moderate salt concentrations. In contrast, chaotropic ions destabilize BSA and kosmotropic ions stabilize BSA at the higher concentrations. An adequate amount of NaCl or NaSCN prevents the destruction of the environment of the binding site in the segment containing Trp 212 in 4 urea solution at pH 7.0.     

Oligonucleotide studies. Optical rotatory dispersion of five homodinucleotides

1. The optical rotatory dispersion (ORD) of five homodinucleotides, ApAp(3'), CpCp(3'), GpGp(3'), IpIp(3') and UpUp(3') (where A, C, G, I and U represent adenosine, cytidine, guanosine, inosine and uridine respectively, and p to the left of a nucleoside symbol indicates a 5'-phosphate and to the right it indicates a 3'-phosphate), were measured as a function of pH, ionic strength and Mg(2+) concentration. 2. The ORD titrations of ApAp(3') and CpCp(3'), which were made by measuring the ORD curves at closely spaced pH intervals, exhibit a maximum at approx. pH5.0 and 5.7 for ApAp(3') and CpCp(3') respectively in the profile of the magnitude of the first Cotton effect versus pH. The results indicate that the conformational rigidity of these dinucleotides depends on the ionization state of a 3'-terminal phosphate group. 3. ApAp(3') was shown to exist as an approximately 1:1 equilibrium mixture of the two major ionic species represented by Ap((-1))Ap((-1)) and Ap((-1))Ap((-2)) at pH6.16, whereas at pH7.5 it exists exclusively as a form of Ap((-1))Ap((-2)). 4. To ascertain the effects of the presence of a terminal phosphate group and of the ionization of the secondary phosphate on the conformation of adenylate dimer, we measured the ORD of ApA, ApAp(3')CH(3) and ApAp(2'). The rotatory power of the first Cotton effect in the above series of dinucleotides decreased at 20 degrees in the order ApA> ApAp(3')CH(3) approximately ApAp(3')((-1))> ApAp(2') at pH7> ApAp(3') at pH7. 5. The pH-rotation profiles were also obtained for ApAp(2'), CpCp(2') and UpUp(3'), but no corresponding maximum was observed. Although simple nearest-neighbour calculations based on the ORD data of IpIp(3') and 5'-IMP account for the observed ORD spectrum of polyinosinic acid at low salt concentration, there were large discrepancies between calculated and experimental results of the polyguanylic acid ORD even at low ionic strength. 6. The extent to which the amplitude of the Cotton effects of IpIp(3') increases with salt concentration, especially by the addition of Mg(2+), was much greater than that observed for ApAp(3'). The implication of such salt effects on the ORD is considered.     

The effect of salt on the Michaelis Menten constant of the HIV-1 protease correlates with the Hofmeister series

The effect of different types of salt on the proteolytic activity of HIV-1 protease was studied. At a similar ionic strength, the enzyme activity changed according to the salting out effect of the ions used (Hofmeister series). Kinetic studies showed that a stronger salting out effect of the ions rather than the higher ionic strength per se increased the affinity to the substrate (Km) but in general did not alter the Kcat value.     

Salt bridge interactions: Stability of the ionic and neutral complexes in the gas phase,in solution,and in proteins

A theoretical study on the stability of the salt bridges in the gas phase, in solution, and in the interior of proteins is presented. The study is mainly focused on the interaction between acetate and methylguanidinium ions, which were used as model compounds for the salt bridge between Asp (Glu) and Arg. Two different solvents (water and chloroform) were used to analyze the effect of varying the dielectric constant of the surrounding media on the salt bridge interaction. Calculations in protein environments were performed by using a set of selected protein crystal structures. In all cases attention was paid to the difference in stability between the ion pair and neutral hydrogen-bonded forms. Comparison of the results determined in the gas phase and in solution allows us to stress the large influence of the environment on the binding process, as well as on the relative stability between the ionic and neutral complexes. The high anisotropy of proteins and the local microenvironment in the interior of proteins make a decisive contribution in modulating the energetics of the salt bridge. In general, the formation of salt bridges in proteins is not particularly favored, with the ion pair structure being preferred over the interaction between neutral species. Proteins 32:67–79, 1998. © 1998 Wiley-Liss, Inc.     

Cation binding linked to a sequence-specific CAP-DNA interaction

The equilibrium association constant observed for many DNA-protein interactions in vitro (K(obs)) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that release of cations from the DNA is the dominant involvement of ions in the binding reaction. With this assumption, the graph of logK(obs) versus log[MX] is predicted to have a constant slope proportional to the number of ions released from the DNA upon protein binding. However, experimental data often deviate from log-linearity at low salt concentrations. Here we show that for the sequence-specific interaction of CAP with its primary site in the lactose promoter, ionic stoichiometries depend strongly on cation identity and weakly on anion identity. This outcome is consistent with a simple linkage model in which cation binding by the protein accompanies its association with DNA. The order of ion affinities deduced from analysis of DNA binding is the same as that inferred from urea-denaturation experiments performed in the absence of DNA, suggesting that ion binding to free CAP contributes significantly to the ionic stoichiometry of DNA binding. In living cells, the coupling of ion-uptake and DNA binding mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.