The binding of monomeric amyloid ß peptide to serum albumin is affected by major plasma unsaturated fatty acids.

Human serum albumin (HSA) serves as a natural depot of amyloid ß peptide (Aß). Improvement of Aß binding to HSA should impede Alzheimer's disease (AD). We developed a method for quantitation of the interaction between monomeric Aß40/42 and HSA using surface plasmon resonance spectroscopy. The dissociation constant of HSA complex with recombinant Aß40/42 is 0.2-0.3 µM. Flemish variant of Aß40 has 2.5-10-fold higher affinity to HSA. The parameters of the HSA-Aß interaction are selectively sensitive to HSA binding of major plasma unsaturated fatty acids and Cu2+. Linoleic and arachidonic acids promote the HSA-Aß42 interaction. The developed methodology for quantitation of HSA-Aß interaction may serve as a tool for search of compounds favoring HSA-Aß interaction, thereby preventing AD progression.

Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.

The residue Asp87, which is in the calcium-binding loop of bovine alpha-lactalbumin (alpha-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A alpha-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648-11654]. On the contrary, D87N alpha-LA displayed native-like secondary structure with a somewhat de-stabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N alpha-LA [Veprintsev et al. (1999) PROTEINS: Struct. Funct. Genet., 37, 65-72], the structure appeared to more closely resemble native alpha-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N alpha-LA was approximately 31 degrees C versus native apo- alpha-LA (approximately 25 degrees C), probably due to negative charge 'compensation' in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N alpha-LA was approximately 57 degrees C versus native alpha-LA (approximately 66 degrees C), which was related to a decreased Ca(II) affinity (K = approximately 2.1 x 10(5) versus approximately 1.7 x 10(7)/M at 40 degrees C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.

alpha-Lactalbumin: structure and function.

Small milk protein alpha-lactalbumin (alpha-LA), a component of lactose synthase, is a simple model Ca(2+) binding protein, which does not belong to the EF-hand proteins, and a classical example of molten globule state. It has a strong Ca(2+) binding site, which binds Mg(2+), Mn(2+), Na(+), and K(+), and several distinct Zn(2+) binding sites. The binding of cations to the Ca(2+) site increases protein stability against action of heat and various denaturing agents, while the binding of Zn(2+) to the Ca(2+)-loaded protein decreases its stability. Functioning of alpha-LA requires its interactions with membranes, proteins, peptides and low molecular weight substrates and products. It was shown that these interactions are modulated by the binding of metal cations. Recently it was found that some folding variants of alpha-LA demonstrate bactericidal activity and some of them cause apoptosis of tumor cells.

Zinc binding in bovine alpha-lactalbumin: sequence homology may not be a predictor of subtle functional features.

alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution.

Zn(2+)-mediated structure formation and compaction of the "natively unfolded" human prothymosin alpha.

Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites.

A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca(2+)-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M(-1) for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.

Fine tuning the N-terminus of a calcium binding protein: alpha-lactalbumin.

The effects of amino acid substitutions in the N-terminus of bovine recombinant alpha-lactalbumin (including enzymatic removal of the N-terminal methionine and deletion of Glu-1) were studied by intrinsic fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC). Wild-type recombinant alpha-lactalbumin has a lower thermostability and calcium affinity compared to the native protein, while the properties of wild-type protein with the N-terminal methionine enzymatically removed are similar to the native protein. Taken together, the fluorescence, CD, and DSC results show that recombinant wild type alpha-lactalbumin in the absence of calcium ion is in a type of molten globule state. The delta-E1 mutant, where the Glu(1)residue of the native sequence is genetically removed, leaving an N-terminal methionine in its place, shows almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. It was concluded that the N-terminus of the protein dramatically affects both stability and function as manifested in calcium affinity. Proteins 1999;37:65-72.

Cooperative thermal transitions of bovine and human apo-alpha-lactalbumins: evidence for a new intermediate state.

The thermal denaturation of bovine and human apo-alpha-lactalbumins at neutral pH has been studied by intrinsic protein fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC) methods. Apo-alpha-lactalbumin possesses a thermal transition with a midpoint about 25-30 degrees C under these conditions (pH 8.1, 10 mM borate, 1 mM EGTA), which is reflected in changes in both fluorescence emission maximum and quantum yield. However, the CD showed a decrease in ellipticity at 270 nm with a midpoint at about 10-15 degrees C, while DSC shows the transition within the region of 15-20 degrees C. The non-coincidence of transition monitored by different methods suggests the existence of an intermediate state in the course of the thermal denaturation process. This intermediate state is not the classical molten globule state which occurs at higher temperature (i.e. denatured state at these conditions) [D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S.Y. Venyaminov and O.B. Ptitsyn, FEBS Letters, 136 (1981) 311-315] and has physical properties intermediate between the native and molten globule states.

pH-induced transition and Zn2+-binding properties of bovine prolactin.

A pH-induced conformational transition was found in bovine prolactin within the physiologically significant pH region from 6.5 to 8.5. The thermal stability of prolactin at pH 6.5 is essentially higher than at pH 8.5. Bovine prolactin binds zinc ions with an apparent association constant of 2 x 10(5) M(-1) at pH 6.5 and 1 x 10(4) M(-1) at pH 8.5. The pH dependence of both thermal stability and zinc binding surrounding the pKa of histidine suggests that these residues plays a key role in the structural integrity of bovine prolactin.

Interactions of alpha-lactalbumin with fatty acids and spin label analogs.

Bovine alpha-lactalbumin (alpha-LA) has been shown by intrinsic protein fluorescence and electron spin resonance methods to interact with the spin-labeled fatty acid analog, 5-doxylstearic acid, as well as stearic acid. An intrinsic fluorescence titration of various alpha-LA forms with 5-doxylstearic acid causes first an increase and then a decrease in emission intensity with concomitant shifts in tryptophan emission wavelength. In some cases, up to three steps in the fluorescence titration curves were visible, which were fit to apparent binding steps from 10(-6) to 10(-4) M. The binding parameters of 5-doxylstearic acid for apo- and Ca2+-alpha-LA were an order of magnitude different from one another; the stronger one, apo-alpha-lactalbumin, exhibited a Kd of 35 microM. Electron spin resonance titrations of 5-doxylstearic acid-loaded apo-alpha-LA with stearate (micelles) seem to suggest separate binding loci if alpha-LA indeed binds stearate at these concentrations. The titration of alpha-LA by stearic acid results in a fluorescence emission red shift and an apparent stepped increase in fluorescence intensity. Lipid-protein association occurred at concentrations at which stearic acid micelles and aggregates begin to form in the absence of protein. Nonetheless, the relatively strong association between stearic acid and apo-alpha-LA was also confirmed by means of the fluorescent indicator acrylodated fatty acid binding protein, in which addition of alpha-LA to the stearate-loaded indicator protein reverses the decrease in fluorescence of the acrylodan chromophore conjugated to the protein.

Study of tyrosine-containing mutants of ribosomal protein L7/L12 from Escherichia coli.

Three mutant forms of the ribosomal protein L7/L12 with replacements of Ser1, Met14 and Met26 to Tyr were studied by the methods of fluorescence spectroscopy, circular dichroism and microcalorimetry. The amino-acid residue Tyr14 in the protein L7/L12 Tyr14 is located in a region with a more organized structure than Tyr26 in protein L7/L12 Tyr26. The replacements Ser1-->Tyr1 and Met14-->Tyr14 do not affect the secondary structure of protein L7/L12. The replacement Met26-->Tyr26 stabilizes the secondary structure of protein L7/L12. A pH-induced temperature transition was observed in the pH range 5.0-7.3 in protein L7/L12 Tyr14 by tyrosine fluorescence. Analogous transitions were observed for protein L7/L12 Tyr26 by Tyr fluorescence and for the wild type protein L7/L12 by Phe fluorescence. Three pH-dependent states of protein L7/L12 and its mutant forms L7/L12 Tyr1 and L7/L12 Tyr14 were found on the microcalorimetric melting curves. The characteristics of protein L7/L12 Tyr14 are very close to the wild type protein L7/L12 and it is a suitable object for studying the structure of the N-terminal part of molecule by two-dimentional 1H-NMR.

Pb2+ and Hg2+ binding to alpha-lactalbumin.

Interactions of human alpha-lactalbumin with Pb2+ and Hg2+ were studied by intrinsic protein fluorescence. Lead ions bind to the strong Ca2+ binding site of alpha-lactalbumin (association constant Kass approximately 2 x 10(6) M-1) with concomitant spectral changes which are similar to those induced by the binding of Ca2+. Pb2+ also binds to the strong Zn2+ site with Kass approximately 10(5) M-1 and some secondary binding site(s) (which probably contain histidine residues) with apparent Kass approximately 10(4) M-1, causing pronounced aggregation of the protein. Mercury ions bind to alpha-lactalbumin at the primary Zn2+ sites with Kass approximately (1-4) x 10(4) M-1, although the stoichiometry of the binding depends on the conformational state of the protein. Secondary Hg2+ binding sites were suggested to contain histidines, while the strong Hg2+ site contains carboxylates in the coordination sphere and seems to coincide with the strong Zn2+ site. The binding of both Pb2+ and Hg2+ decreases the thermal stability of the Ca(2+)-loaded protein and in some conditions causes pronounced protein aggregation.

Interactions of alpha-lactalbumins with lipid vesicles studied by tryptophan fluorescence.

Complexes of alpha-lactalbumin (alpha-LA)1 with dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes at pH 8 and at pH 2 have been obtained by means of gel filtration. Thermal denaturation of alpha-LA complexes of DMPC or DPPC at pH 8 was found to depend on the saturation of protein by metal cations. The intrinsic fluorescence of DMPC-alpha-LA and DPPC-alpha-LA was sensitive to two thermal transitions. The first transition corresponded to the Tc of the lipid vesicles, while the second transition arose from the denaturation of the protein. Fluorescence spectrum position suggested that at low temperature tryptophan accessibility increases upon protein-DMPC or protein-DPPC association. At temperatures above the protein transition (70 degrees C) tryptophan appears to interact significantly with the apolar phase of DMPC and DPPC, evidenced by spectral blue shifts. Whereas the free protein at pH 2 adopts the molten globule (MG) state and is characterized by the absence of a thermal transition, the rapidly-isolated DMPC-alpha-LA complex was characterized by the appearance of a distinct fluorescence thermal transition between 50 and 60 degrees C. This result is consistent with a model of a partially-inserted form of alpha-LA which may possess some degree of tertiary structure and therefore unfolds cooperatively.

Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa.

The proteins, AlgR3 and AlgP, are involved in the regulation of alginate synthesis in Pseudomonas. They contain multiple repeats of Ala*Ala*Lys*Pro as do several other proteins that resemble histones. The interactions of synthesis oligopeptides composed of repeated Ala*Ala*Lys*Pro or Lys*Lys*Ser*Pro units with DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl) group attached to the N-termini of the peptides. DNA quenching of the Fmoc fluorescence of the peptides was used to estimate the apparent association constants for the interaction of Fmoc(AAKP)nOH (n = 2, 4, 8, 18, 32) and of Fmoc (KKSP)nOH (n = 2, 4, 8, 16, 20, 32) with DNA. The Fmoc(AAKP)nOH peptides bind to DNA only at low ionic strength; the Fmoc(KKSP)n OH peptides interact with DNA at both low (0.05 M KCl) and high (0.2 M KCl) salt. At low ionic strength an increase in the number of the repeat units causes an increase in the apparent association constant up to approximately 2 x 10(6) M-1 for both types of peptides at N congruent to 24. The insertion of an AAKTA unit into the middle of the Fmoc(AAKP)8OH peptide increases its affinity to DNA. We propose a model of (AAKP)n and of its interaction with DNA. The repeat unit consists of a single turn of alpha-helix followed by a bend necessitated by Pro. The resultant coiled-coil forms a right-handed superhelix with 10 AAKPs per repeat distance of approximately 33 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of peptide synthesis studied by fluorescence of fluorophenyl esters.

The kinetics of the reaction of Boc-alanine-trifluorophenyl, Boc-alanine-tetrafluorophenyl, Boc-alanine-pentafluorophenyl, and Boc-alanine-p-chlorotetrafluorophenyl esters (BocAlaOTrf, BocAlaOTfp, BocAlaOPfp, and BocAlaTfc, respectively) with leucine amide and with valine methyl ester have been measured using changes in fluorophenyl chromophore emission at 375 nm. The kinetic data cannot be well fit with a simple second-order reaction scheme. Measurements of the reaction kinetics at different concentrations of the reagents showed that the expression for the reaction rate is V = kC(N)0.5C(AE)1.5, in which k is the reaction rate constant, CN is the concentration of either LeuNH2 or ValOCH3, and CAE is the concentration of the fluorophenyl ester. This reaction equation indicates a complex, probably chain-like, reaction mechanism. The order of reactivity for these active esters with ValOCH3 is BocAlaOTfc > BocAlaOPfp > BocAlaOTfp > BocAlaTrf. The apparent rate constant, k, for the reaction with LeuNH2 is higher than that for the reaction with ValOCH3.

Co2+ binding to alpha-lactalbumin.

alpha-Lactalbumin possesses multiple Zn2+ binding sites, with the strongest site having an affinity constant of 5 x 10(5) M-1 [Permyakov et al. (1991), J. Protein Chem. 100, 577]. The binding of zinc at secondary sites is accompanied by destabilization of the protein structure and progressive protein aggregation. This pronounced destabilization is reflected in a shift of the thermal denaturation transition temperature by more than 40 degrees. The present work examines Co2+ binding to bovine alpha-lactalbumin, where for this analog of Zn2+, multiple binding sites were also found from spectrofluorimetric titrations. The strong site Co2+ binding constant was 1.3 x 10(6) M-1. However, in contrast to Zn2+ binding, Co2+ does not cause protein aggregation nor any significant thermal destabilization of the protein. Fluorescence energy transfer measurements between Tb3+ in the strong calcium site to Co2+ in the strong Zn2+ site gave a distance in the range of 14-18 A, which was in excellent agreement with recent crystallographic data for human alpha-lactalbumin [Ren et al. (1993), J. Biol. Chem. 268, 19292-19298]. However, the X-ray structure did not identify the additional zinc sites found from earlier solution studies, presumably due to restrictive crystal packing interactions. The results from the current work confirm that the strong cobalt (zinc) site in solution is the same zinc site elucidated by X-ray crystallography.

Effects of Zn(II) on galactosyltransferase activity.

The enzyme beta-4-galactosyltransferase (GT) catalyzes the transfer of a galactosyl group from UDP-galactose to N-acetylglucosamine (GlcNAc) on glycoproteins. In the presence of alpha-lactalbumin (alpha-LA), galactosyltransferase catalyzes the transfer of galactose to glucose to yield lactose. It is known that, in the absence of alpha-lactalbumin, Zn(II) competes with Mn(II) for the same binding site(s) in galactosyltransferase, resulting in an increase in the apparent Michaelis constant, Km(app), for Mn(II)-activation of N-acetyllactosamine synthesis. In the presence of alpha-lactalbumin (i.e., lactose synthase), the Mn(II)-activation is biphasic and the initial phase is inhibited by increasing concentrations of Zn(II). The Zn(II) inhibition of lactose synthase plateaus at [Zn(II)]:[alpha-lactalbumin] approximately 1:1, while for N-acetyllactosamine synthesis there is no plateau at all. The results suggest that Zn(II) binding to alpha-lactalbumin effects lactose synthase. Kinetically, Zn(II) induces a decrease in both the Km(app) and Vm for Mn(II), which results in an apparent increase, followed by a decrease, in lactose synthase activity at Mn(II) concentrations below saturation of the first [Mn(II)] binding site. Increasing Zn(II) also decreases Km(app) and Vm for both glucose and UDP-galactose in the lactose synthase reaction with either both Ca(II)- or apo-alpha-lactalbumin, further suggesting novel interactions between Zn(II)-alpha-lactalbumin and the lactose synthase complex, presumably mediated via a Zn(II)-induced conformational change upon binding to alpha-lactalbumin. On the other hand, in N-acetyllactosamine synthesis, Zn(II) only slightly effects Km(app) for N-acetylglucosamine and has essentially no effect on Km(app) or Vm for UDP-galactose.

Introduction of Ca(2+)-binding amino-acid sequence into the T4 lysozyme.

The 51-62 loop of T4 phage lysozyme was altered by site-directed mutagenesis to obtain maximal homology with the typical EF-hand motif. A Ca(2+)-binding site was designed and created by replacing both Gly-51 and Asn-53 with aspartic acid. The mutant T4 lysozyme (G51D/N53D) was expressed in Escherichia coli. The activity of the G51D/N53D-mutant was about 60% of that of the wild-type protein. This mutant can bind Ca2+ ions specifically, while the effective dissociation constant was essentially greater than that of the EF-hand proteins. Stability of the G51D/N53D-mutant apo-form to urea- or temperature-induced denaturation was the same as that of the wild-type protein. In the presence of Ca2+ ions in solution the stability of the mutant T4 phage lysozyme was less than that of the wild-type protein. It is suggested that the binding of Ca2+ by the mutant is accompanied by the considerable conformational changes in the 'corrected' loop, which can lead to the Ca(2+)-induced destabilization of the protein.

Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1.

The thermal unfolding and domain structure of myosin subfragment 1 (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 degrees C), while the binding of the non-hydrolysable analogue of ATP, adenosine 5'-[beta, gamma-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1 degrees C, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide (pPDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-Vi can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-Pi.

Interaction of cupric ion with parvalbumin.

Cod parvalbumin, a calcium-binding protein, possesses a specific Zn2+ (or Cu2+) binding site per molecule. This work employed fluorescence energy transfer techniques to measure the distance between the Zn2+ (Cu2+) site and the stronger Ca(2+)-binding site in parvalbumin. Specifically, the distance between Tb3+ bound at the Ca2+ site and Co2+ bound to the Zn2+ (Cu2+) binding site was 10.3 +/- 0.9 A. Lastly, the effects of Cu2+ on the physico-chemical properties of parvalbumin were studied by measuring the accessibility of protein thiol groups to 5,5'-dithio bis(2-nitrobenzoic acid) and by its affinity for the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulfonic acid] dipotassium salt. The thiol group accessibility decreased and the affinity to the fluorescent probe increased upon complexation of Cu2+ to the protein. It appears that the binding of Cu2+ converts parvalbumin to an apo-like state.