Function of the F1-motor (F1-ATPase) of ATP synthase by apolar-polar repulsion through internal interfacial water

THESIS: Within the structurally-confined internal aqueous cavity of the F1-motor of ATP synthase, function results from free energy changes that shift the balance between interfacial charge hydration and interfacial hydrophobic hydration. TRANSITION STATE DESCRIPTION: At the beta-P end of ADP x Mg occurs an inorganic phosphate, P(i). This P(i) resides at the base of a water-filled cleft that functions like an aperture to focus, into an aqueous chamber, a competition for hydration (an apolar-polar repulsion) between charged phosphate and hydrophobic surface of the gamma-rotor. Two means available for the phosphate and the hydrophobic surface to improve their hydration free energies are physically to separate by rotation of the gamma-rotor or chemically to combine P(i) with ADP to form less charged ATP. This proposal derives from calculated changes in Gibbs free energy for hydrophobic association of amino acid side chains and chemical modifications thereof and from experimentally demonstrated water-mediated repulsion between hydrophobic and charged sites that resulted from extensive studies on designed elastic-contractile model proteins.     

Combined effect of hydrodynamic and interfacial flow parameters on lysozyme deactivation in a stirred tank bioreactor

The dynamic environment within a bioreactor and in the purification equipment is known to affect the activity and yield of enzyme production. The present research focuses on the effect of hydrodynamic flow parameters (average energy dissipation rate, maximum energy dissipation rate, average shear rate, and average normal stress) and the interfacial flow parameters (specific interfacial area and mass transfer coefficient) on the activity of lysozyme. Flow parameters were estimated using CFD simulation based on the k-epsilon approach. Enzyme deactivation was investigated in 0.1, 0.3, 0.57, and 1 m i.d. vessels. Enzyme solution was subjected to hydrodynamic stress using various types of impellers and impeller combinations over a wide range of power consumption (0.03 < P(G)/V < 7, kW/m3). The effects of tank diameter, impeller diameter, blade width, blade angle, and the number of blades on the extent of deactivation were investigated. At equal value of P(G)/V, epsilon(max), and gamma(avg), the extent of deactivation was dramatically different for different impeller types. The extent of deactivation was found to correlate well with the average turbulent normal stress and the mass transfer coefficient.     

Membrane fluidity gradient model of cell transport

A new model of cellular transport is presented, characterized by selective fluxes due to membrane fluidity gradient. This mechanism is treated in terms of the interfacial tensions at the membrane/cytoplasm and membrane/medium surfaces. A higher interior fluidity (lower interfacial tension) is maintained by cytoplasm adenosine triphosphate, which adsorbs and increases lipoprotein fluidity while it also chelates calcium and keeps it from inner membrane sites. The high medium calcium causes a stiffer membrane (higher interfacial tension) on the medium side. These two different free energy barriers at inner and outer channel mouths filter all molecules, whether ionized or nonelectrolytic. Molecules with excess of hydrophobic groups, which makes negative the free energy of transfer from the medium into the membrane, have highest influx. Intermolecular salt linkages and hydrogen-bonding are vital in making negative the free energy of transfer of amino acids and sugars. The much lower energy barrier at the cytoplasmic interface favors net efflux from the cell of the more polar ions and amphipaths. Intramembrane particles are proposed as the channel sites.     

What is the Binding Energy of a Charge Transfer State in an Organic Solar Cell?

The high efficiencies reported for organic solar cells and an almost negligible thermal activation measured for the photogeneration of charge carriers have called into question whether photoinduced interfacial charge transfer states are bound by a significant coulomb attraction, and how this can be reconciled with very low activation energies. Here, this question is addressed in a combined experimental and theoretical approach. The interfacial binding energy of a charge‐transfer state in a blend of MeLPPP:PCBM is determined by using energy resolved electrochemical impedance spectroscopy and is found to be about 0.5 eV. Temperature‐dependent photocurrent measurements on the same films, however, give an activation energy that is about one order of magnitude lower. Using analytical calculations and Monte Carlo simulation the authors illustrate how i) interfacial energetics and ii) transport topology reduce the activation energy required to separate the interfacial electron–hole pair, with about equal contributions from both effects. The activation energy, however, is not reduced by entropy, although entropy increases the overall photodissociation yield.     

Interfacial Tension and Surface Pressure of High Density Lipoprotein,Low Density Lipoprotein,and Related Lipid Droplets

Lipid droplets play a central role in energy storage and metabolism on a cellular scale. Their core is comprised of hydrophobic lipids covered by a surface region consisting of amphiphilic lipids and proteins. For example, high and low density lipoproteins (HDL and LDL, respectively) are essentially lipid droplets surrounded by specific proteins, their main function being to transport cholesterol. Interfacial tension and surface pressure of these particles are of great interest because they are related to the shape and the stability of the droplets and to protein adsorption at the interface. Here we use coarse-grained molecular-dynamics simulations to consider a number of related issues by calculating the interfacial tension in protein-free lipid droplets, and in HDL and LDL particles mimicking physiological conditions. First, our results suggest that the curvature dependence of interfacial tension becomes significant for particles with a radius of ～5 nm, when the area per molecule in the surface region is <1.4 nm². Further, interfacial tensions in the used HDL and LDL models are essentially unaffected by single apo-proteins at the surface. Finally, interfacial tensions of lipoproteins are higher than in thermodynamically stable droplets, suggesting that HDL and LDL are kinetically trapped into a metastable state.     

On liquid-liquid mass transfer in two-liquid-phase fermentations

Almost all two-liquid phase bioprocesses are characterized by the presence of surface active materials (biosurfactants), which significantly influence the interaction between the phases. In order to predict mass transfer rates during cultivations of Pseudomonas oleovorans biosurfactant was isolated from the biosuspension and added in defined amounts to n-octane/water model-dispersions. Effects of biosurfactant concentration on interfacial tension, mean Sauter-diameter, drop size distribution, dispersion stability and liquid-liquid mass transfer coefficients were studied. A comparison was made between calculated solvent transfer rates (STR) and measured solvent uptake rates (SUR) of P. oleovorans cultures. With increasing interfacial surfactant concentration interfacial tension and mean Sauter-diameter decreased until a minimum for both, interfacial tension and mean Sauter-diameter, were reached. Interfacial tension measurements indicate that these minima have to be attributed to a maximum monomolecular surfactant concentration and the formation of polymolecular adsorption layers. Drop size distributions showed that, coalescence and droplet break-up disappear because droplets are stabilized by the biosurfactant adsorption layers at the interface. Mass transfer regime shifted from forced convection and surface renewal to diffusion. Comparison of solvent uptake rates (SUR) and solvent transfer rates (STR) showed that n-octane transfer usually will not be limiting P. oleovorans cultures, however, can become dominant in cultures where solvents with very low miscibilities like n-decane are used.     

Surface and aggregate properties of an amphiphilic derivative of carboxymethylchitosan

A new kind of amphiphilic derivative of carboxymethylchitosan, a group of (2-hydroxyl-3-butoxyl)propylcarboxymethylchitosans (HBP-CMCHS), has been synthesized, and the surface and aggregate properties have been studied by means of surface tension, surface pressure and fluorescence measurements. HBP-CMCHS can adsorb on the surface to decrease the surface tension of the solution. The adsorption film was quite stable, which can make the relative compressed pressure increase dramatically with the decrease of the surface area. In solution, hydrophobic aggregations were identified by the decrease in the ratio of the fluorescence emission intensity of the first and third pyrene vibronic peaks ( I(1)/ I(3)). Results showed that the aggregation began to form at a concentration similar to that of the polymer transfer to the air-water interface. Aggregate formation of the polymers is a gradually compact process with hydrophobic associations. Increase of DS and addition of NaCl to the HBP-CMCHS solution can make the surface tension decrease, make the aggregation occur at lower concentration, and make the aggregation more hydrophobic.     

Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane

Co-translational translocation of proteins across the membrane of rough endoplasmic reticulum (ER) is interrupted by particular amino acid sequences, which are functionally termed "stop-transfer sequence." We analyzed the structural requirements for the interruption of the peptide translocation. By the manipulation of the cDNA of interleukin 2 (IL2), which passes through ER membrane co-translationally, the middle portion of the IL2 molecule was replaced with systematically altered hydrophobic segments, leucine, alanine, or leucine/alanine mixed clusters. Furthermore, charged amino acid residues were introduced just downstream of the hydrophobic segments. These modified IL2 peptides were synthesized with wheat germ cell-free system in the presence of rough microsomes and the topology of the peptides in the microsomes was assessed by post-translational digestion with proteinase K. We obtained the following results. (i) Each modified protein was processed to the mature form but the extent of stop-translocation varied widely. The ratio of the stopped to the translocated products increased as the length and hydrophobicity of the inserted segment increased. (ii) Shorter hydrophobic segments than naturally occurring native transmembrane segment promoted stop-translocation. (iii) Proteins with hydrophobic segments followed by positive charges were more efficiently stop-translocated than those having negative charges. (iv) If the hydrophobicity of the segment was sufficiently high, the positive charges after the segment were not essential for stop-translocation. We also suggest that the stop-transfer process includes protein-protein interaction between the hydrophobic segment and translocation channel.     

Simian virus 40-transformed cells express new species of proteins precipitable by anti-simian virus 40 tumor serum.

In addition to the virus-coded large-T and small-t antigens, two new classes of proteins were immunoprecipitated by anti-simian virus 40 (SV40) tumor serum from extracts of various SV40-transformed cell lines. These were as follows: (i) proteins (termed "super-T proteins") with an Mr higher than that of large-T antigen (86,000), which were found in many SV40-transformed cell lines derived from mouse and rat cells (super-T proteins and large-T antigen appeared to have closely related structures as judged by the Chromobead elution patterns of their methionine-labeled tryptic peptides); (ii) proteins (termed "55K proteins") with an Mr ranging from 50,000 to 60,000, which were present in all SV40-transformed cell lines examined so far, including those obtained by chromosome-mediated gene transfer. The 55K proteins were not structurally related to large-T antigens, as judged by the Chromobead elution patterns of their methionine-labeled tryptic peptides. Our data are compatible with the assumption that the 55K proteins are largely or totally cell coded.     

A model of the quinone-binding site of photosystem I: investigation of charge transfer complexes of phylloquinone and its derivatives with tryptophan

Phylloquinone (vitamin K1; 2-methyl-3-phytyl-1,4-naphthoquinone), the secondary electron acceptor A1 in photosystem I of plants, algae, and cyanobacteria, mediates the electron transfer between A0 (a monomeric chlorophyll a) and the iron-sulfur cluster Fx. In order to investigate the interaction of vitamin K1 with the A1-binding site, their models on the non-covalent complexes were obtained and studied. The fluorescent properties of vitamin K1, its derivatives, and their complexes with Trp showed a possible role of amino acid components in the formation of a stable energy state, which provides the energy redistribution between oxidized and reduced forms of vitamin K1. The formation of the charge-transfer complex and the influence of the ratio of the components on fluorescence derived from the tryptophan-vitamin K1, tryptophan-dihydrovitamin K1 , tryptophan-quinhydrone K1, and tryptophan-naphthochromanol complexes are described. The data obtained allow us to suggest that naphthochromanol is involved in the energy transfer reaction in PS I as an intermediate form of the secondary electron acceptor.     

Differential activation of stress-responsive signalling proteins associated with altered loading in a rat skeletal muscle

Skeletal muscle undergoes a significant reduction in tension upon unloading. To explore intracellular signalling mechanisms underlying this phenomenon, we investigated twitch tension, the ratio of actin/myosin filaments, and activities of key signalling molecules in rat soleus muscle during a 3-week hindlimb suspension and 2-week reloading. Twitch tension and myofilament ratio (actin/myosin) gradually decreased during unloading but progressively recovered to initial levels during reloading. To study the involvement of stress-responsive signalling proteins during these changes, the activities of protein kinase C alpha (PKCalpha) and three mitogen-activated protein kinases (MAPKs)--c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 MAPK--were examined using immunoblotting and immune complex kinase assays. PKCalpha phosphorylation correlated positively with the tension (Pearson's r = 0.97, P < 0.001) and the myofilament ratio (r = 0.83, P < 0.01) over the entire unloading and reloading period. Treatment of the soleus muscle with a PKC activator resulted in a similar paralleled increment in both PKCalpha phosphorylation and the alpha-sarcomeric actin expression. The three MAPKs differed in the pattern of activation in that JNK activity peaked only for the first hours of reloading, whereas ERK and p38 MAPK activities remained elevated during reloading. These results suggest that PKCalpha may play a pivotal role in converting loading stress to intracellular changes in contractile proteins that determine muscle tension. Differential activation of MAPKs may also help alleviate muscle damage, modulate energy transport and/or regulate the expression of contractile proteins upon altered loading.     

Measurement of thermodynamic parameters for hydrophobic mismatch 2: intermembrane transfer of a transmembrane helix

Hydrophobic matching between proteins and lipids is essential for the thermodynamic stability of integral membrane proteins. However, there is no direct thermodynamic information available about the intermembrane transfer of proteins between membranes with different hydrophobic thicknesses, which is crucial for understanding hydrophobic mismatch. This article reports the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the intermembrane transfer of the inert hydrophobic model transmembrane helix NBD-(AALALAA)(3)-NH(2) (NBD: 7-nitro-2-1,3-benzoxadiazol-4-yl), which is exchangeable between vesicles, from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to dimonounsaturated-phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 37-58 degrees C. The transfer free energies were calculated from equilibrium values of the extent of helix transfer from donor to acceptor lipid vesicles, as monitored by a decrease in fluorescence resonance energy transfer from the NBD group to a lipid-labeled Rhodamine in the donor upon transfer to the quencher-free acceptor. Under hydrophobic mismatch conditions up to approximately 7 A, the helix partitioning became unfavorable up to +7 kJ mol(-)(1), hampered by an increase in entropic (up to +20 kJ mol(-)(1)) and enthalpic (up to +66 kJ mol(-)(1)) terms in thinner and thicker membranes, respectively. Together with the results that H/D exchange at the membrane interface was accelerated in thinner membranes the obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, resulting in excess energies originating from the hydration of terminal hydrophobic residues or the unfavorable Born energy of terminal partial charges of the helix macrodipole.     

Binding energy and catalysis by D-xylose isomerase: kinetic, product, and X-ray crystallographic analysis of enzyme-catalyzed isomerization of (R)-glyceraldehyde

D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and d-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D(2)O was monitored by (1)H nuclear magnetic resonance spectroscopy, and a k(cat)/K(m) of 0.034 M(-1) s(-1) was determined for this isomerization at pD 7.0. This is similar to the k(cat)/K(m) of 0.017 M(-1) s(-1) for the TIM-catalyzed carbon deprotonation reaction of DGA in D(2)O at pD 7.0 [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (k(cat)/K(m) = 490 M(-1) s(-1)) versus that for the TIM-catalyzed isomerization of DGAP (k(cat)/K(m) = 9.6 × 10(6) M(-1) s(-1)) is due to (i) the barrier to conversion of cyclic d-xylose to the reactive linear sugar (5.4 kcal/mol) being larger than that for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol) and (ii) the intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219-410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) being smaller than that of the phosphodianion group of DGAP (~12 kcal/mol). The XI-catalyzed isomerization of DGA in D(2)O at pD 7.0 gives a 90% yield of [1-(1)H]DHA and a 10% yield of [1-(2)H]DHA, the product of isomerization with incorporation of deuterium from solvent D(2)O. By comparison, the transfer of (3)H from the labeled hexose substrate to solvent is observed only once in every 10(9) turnovers for the XI-catalyzed isomerization of [2-(3)H]glucose in H(2)O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994) Biochemistry 33, 1481-1487]. We propose that truncation of the terminal ethylene glycol fragment of d-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that for proton transfer. An ultra-high-resolution (0.97 ?) X-ray crystal structure was determined for the complex obtained by soaking crystals of XI with 50 mM DGA. The triose binds to XI as the unreactive hydrate, but ligand binding induces metal cofactor movement and conformational changes in active site residues similar to those observed for XI·sugar complexes.     

Energetics of inclusion-induced bilayer deformations.

The material properties of lipid bilayers can affect membrane protein function whenever conformational changes in the membrane-spanning proteins perturb the structure of the surrounding bilayer. This coupling between the protein and the bilayer arises from hydrophobic interactions between the protein and the bilayer. We analyze the free energy cost associated with a hydrophobic mismatch, i.e., a difference between the length of the protein's hydrophobic exterior surface and the average thickness of the bilayer's hydrophobic core, using a (liquid-crystal) elastic model of bilayer deformations. The free energy of the deformation is described as the sum of three contributions: compression-expansion, splay-distortion, and surface tension. When evaluating the interdependence among the energy components, one modulus renormalizes the other: e.g., a change in the compression-expansion modulus affects not only the compression-expansion energy but also the splay-distortion energy. The surface tension contribution always is negligible in thin solvent-free bilayers. When evaluating the energy per unit distance (away from the inclusion), the splay-distortion component dominates close to the bilayer/inclusion boundary, whereas the compression-expansion component is more prominent further away from the boundary. Despite this complexity, the bilayer deformation energy in many cases can be described by a linear spring formalism. The results show that, for a protein embedded in a membrane with an initial hydrophobic mismatch of only 1 A, an increase in hydrophobic mismatch to 1.3 A can increase the Boltzmann factor (the equilibrium distribution for protein conformation) 10-fold due to the elastic properties of the bilayer.     

Properties of integral membrane protein structures: derivation of an implicit membrane potential

Distributions of each amino acid in the trans-membrane domain were calculated as a function of the membrane normal using all currently available alpha-helical membrane protein structures with resolutions better than 4 A. The results were compared with previous sequence- and structure-based analyses. Calculation of the average hydrophobicity along the membrane normal demonstrated that the protein surface in the membrane domain is in fact much more hydrophobic than the protein core. While hydrophobic residues dominate the membrane domain, the interfacial regions of membrane proteins were found to be abundant in the small residues glycine, alanine, and serine, consistent with previous studies on membrane protein packing. Charged residues displayed nonsymmetric distributions with a preference for the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Potentials of mean force along the membrane normal were derived for each amino acid by fitting Gaussian functions to the residue distributions. The individual potentials agree well with experimental and theoretical considerations. The resulting implicit membrane potential was tested on various membrane proteins as well as single trans-membrane alpha-helices. All membrane proteins were found to be at an energy minimum when correctly inserted into the membrane. For alpha-helices both interfacial (i.e. surface bound) and inserted configurations were found to correspond to energy minima. The results demonstrate that the use of trans-membrane amino acid distributions to derive an implicit membrane representation yields meaningful residue potentials.     

The Influence of pH on Phosphatidylethanolamine Monolayer at the Air/Aqueous Solution Interface

The dependence of the interfacial tension of a phosphatidylethanolamine (PE) monolayer on the pH of the aqueous solution has been studied. A theoretical equation is derived to describe this dependence. A simple model of the influence of pH on the phosphatidylethanolamine monolayer at the air/hydrophobic chains of PE is presented. The contributions of additive phosphatidylethanolamine forms (both interfacial tension values and molecular area values) depend on pH. The interfacial tension values and the molecular area values for PEH⁺ and PEOH^? forms of phosphatidylethanolamine were calculated. The assumed model was verified experimentally. The experimental results agreed with those derived from the theoretical equation in a whole range of pH values.     

Differentiated analysis of the secondary structure of hydrophilic and hydrophobic regions in alpha- and beta-subunits of Na+,K+-ATPase by Raman spectroscopy

Raman spectra of active Na+,K+-ATPase from pig kidney and membrane-bound products of its two-stage trypsinolysis, including alpha-subunit hydrophobic regions as well as the intact beta-subunit and hydrophobic regions of alpha- and beta-subunits, were measured to calculate the secondary structure of hydrophilic and hydrophobic regions of the enzyme. Consequent comparison demonstrated unambiguously that (i) membrane-bound hydrophobic parts of polypeptide chains of Na+,K+-ATPase subunits are in the alpha-helical conformation; (ii) essential contents of the alpha-helix as well as beta-sheet are estimated to form the hydrophilic (mainly cytoplasmic) domain of the Na+,K+-ATPase alpha-subunit; (iii) the exoplasmic hydrophilic domain of the beta-subunit is shown to include several antiparallel beta-pleated sheets and a small amount of the alpha-helix and unordered conformations. The model of the secondary structure organization of hydrophilic domains as well as 8 hydrophobic transmembrane segments of the enzyme molecule was proposed on the basis of experimental results and predictional calculations.     

The role of contact sites between inner and outer mitochondrial membrane in energy transfer

Three functions have been suggested to be localized in contact sites between the inner and the outer membrane of mitochondria from mammalian cells: (i) transfer of energy from matrix to cytosol through the action of peripheral kinases; (ii) import of mitochondrial precursor proteins; and (iii) transfer of lipids between outer and inner membrane. In the contact site-related energy transfer a number of kinases localized in the periphery of the mitochondrion play a crucial role. Two examples of such kinases are relevant here: (i) hexokinase isoenzyme I which is capable of binding to the outer aspect of the outer membrane; and (ii) the mitochondrial isoenzyme of creatine kinase which is localized in the intermembrane space. Recently, evidence was presented that both hexokinase and creatine kinase are preferentially localized in contact sites (Adams, V. et al. (1989) Biochim. Biophys. Acta 981, 213-225). The aim of the present experiments was two-fold. First, to establish methods which enable the bioenergetic aspects of energy transfer mediated by kinases in contact sites to be measured. In these experiments emphasis was on hexokinase, while 31P-NMR was the major experimental technique. Second, we wanted to develop methods which can give insight into factors playing a role in the formation of contact sites involved in energy transfer. In the latter approach, mitochondrial creatine kinase was studied using monolayer techniques.     

Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae

The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V(max)/K(M) ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1,10-phenanthroline having a K(i) value of 0.12 mM and cysteine with K(i) value of 0.31 mM, while EGTA stimulated LAP activity. Zn(2+), Mg(2+) and Mn(2+) all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases.     

Ca2+ differently affects hydrophobic properties of guanylyl cyclase-activating proteins (GCAPs) and recoverin

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.