Assembly characteristics of flagellar cap protein HAP2 of Salmonella: decamer and pentamer in the pH-sensitive equilibrium

The folding of a 98 residue protein, muscle acylphosphatase (AcP), has been studied using a variety of techniques including circular dichroism, fluorescence and NMR spectroscopy following transfer of chemically denatured protein into refolding conditions. A low-amplitude phase, detected in concurrence with the main kinetic phase, corresponds to the folding of a minor population (13%) of molecules with one or both proline residues in a cis conformation, as shown from the sensitivity of its rate to peptidyl prolyl isomerase. The major phase of folding has the same kinetic characteristics regardless of the technique employed to monitor it. The plots of the natural logarithms of folding and unfolding rate constants versus urea concentration are linear over a broad range of urea concentrations. Moreover, the initial state formed rapidly after the initiation of refolding is highly unstructured, having a similar circular dichroism, intrinsic fluorescence and NMR spectrum as the protein denatured at high concentrations of urea. All these results indicate that AcP folds in a two-state manner without the accumulation of intermediates. Despite this, the folding of the protein is extremely slow. The rate constant of the major phase of folding in water, kfH2O, is 0.23 s-1 at 28 degreesC and, at urea concentrations above 1 M, the folding process is slower than the cis-trans proline isomerisation step. The slow refolding of this protein is therefore not the consequence of populated intermediates that can act as kinetic traps, but arises from a large intrinsic barrier in the folding reaction.     

Dissociation of Ca2+ from sarcoplasmic reticulum Ca2+-ATPase and changes in fluorescence of optically selected Trp residues. Effects of KCl and NaCl and implications for substeps in Ca2+ dissociation

Sequential dissociation of the two Ca2+ ions bound to non-phosphorylated sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe. The kinetics of Ca2+ dissociation were deduced from the observed changes in quin2 fluorescence in the visible region (with lambdaex = 313 nm), while fluorescence detection in the UV region (with lambdaex = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying this dissociation under the same ionic conditions. In the absence of KCl or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence were monoexponential, with rate constants very close to those of the changes in ATPase tryptophan fluorescence, which also appeared monophasic. In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovalent ions but distinctly slower than the changes in tryptophan fluorescence, which were accelerated to a larger extent. In addition, the apparent kinetics of the Trp fluorescence changes depended on the excitation wavelength. Using an excitation wavelength of 296 nm, the Trp fluorescence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitation wavelength of 290 nm, the rapid drop in tryptophan fluorescence mainly monitors the dissociation of the first of the two Ca2+ ions to be released from Ca2+-ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived ATPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analysis) is faster than the true rate of dissociation of the first Ca2+ ion and in a subsequent slower rise related to dissociation of the second Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of these cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fluorescence level and significantly affects the cooperativity of Ca2+ binding at equilibrium.     

Ca2+-dependent structural changes in C-type mannose-binding proteins

C-type animal lectins are a diverse family of proteins which mediate cell-surface carbohydrate-recognition events through a conserved carbohydrate-recognition domain (CRD). Most members of this family possess a carbohydrate-binding activity that depends strictly on the binding of Ca2+ at two sites, designated 1 and 2, in the CRD. The structural transitions associated with Ca2+ binding in C-type lectins have been investigated by determining high-resolution crystal structures of rat serum mannose-binding protein (MBP) bound to one Ho3+ in place of Ca2+, and the apo form of rat liver MBP. The removal of Ca2+ does not affect the core structure of the CRD, but dramatic conformational changes occur in the loops. The most significant structural change in the absence of Ca2+ is the isomerization of a cis-peptide bond preceding a conserved proline residue in Ca2+ site 2. This bond adopts the cis conformation in all Ca2+-bound structures, whereas both cis and trans conformations are observed in the absence of Ca2+. The pattern of structural changes in the three loops that interact with Ca2+ is dictated in large part by the conformation of the prolyl peptide bond. The highly conserved nature of Ca2+ site 2 suggests that the transitions observed in MBPs are general features of Ca2+ binding in C-type lectins.     

Characterization of the single tyrosine containing troponin C from lungfish white muscle. Comparison with several fast skeletal muscle troponin C's from fish species

Troponin C molecules from fast skeletal muscle of the following fish species (trout, whiting, lungfish, tilapia, and cod) have been purified to homogeneity. Upon binding of Ca2+ or Mg2+, lungfish troponin C is the only troponin C from fish white muscle to show the typical increase of tyrosine fluorescence emission quantum yield reported for rabbit fast skeletal muscle troponin C. The increase of tyrosine fluorescence signal occurring upon Ca2+ and Mg2+ titration of lungfish troponin C has been used to determine the corresponding affinity constants. With K(Ca) = 7.0 10(7) M-1 and K(Mg) = 3.6 10(3) M-1, the sites probed by the tyrosine residue of lungfish troponin C are typical of the COOH-terminal domain of fast skeletal troponin C's. The amino acid sequencing of the tyrosine containing tryptic peptides has allowed us to position the single tyrosine residue at position 7 in the Ca2+ binding loop of the third site, in identical position to Tyr109 of troponin C from rabbit fast skeletal muscle. Metal ion binding studies followed by intrinsic fluorescence or Tb3+ luminescence indicate that the conformation of the structural domain of lungfish troponin C with one metal ion bound is close to the physiological conformation of this domain.     

Characterization of lanthanide ion binding to the EF-hand protein S100 beta by luminescence spectroscopy

S100 beta is a member of a group of low-molecular weight acidic calcium binding proteins widely distributed in the vertebrate nervous system containing two helix-loop-helix calcium binding motifs (sites I and II). In addition, S100 beta also has auxiliary Zn2+ binding sites that are distinct from the Ca2+ binding sites. Luminescence spectroscopy using Eu3+ and Tb3+ as spectroscopic probes for Ca2+ is used to characterize the Ca2+ binding sites of this protein. Eu3+-bound S100 beta shows two distinct Eu3+ binding environments from both the excitation spectrum and Eu3+ excited state lifetimes. Eu3+ bound to the classical EF hand site II has a Kd of 660 +/- 20 nM, whereas the dissociation constant for the pseudo-EF hand site I is significantly weaker. Lifetimes in H2O and D2O lead to the finding that there are four water molecules coordinated to the Eu3+ in the weakly binding site I and two water molecules to the tightly binding site II. Site II in S100 beta expectedly is very similar to high-affinity Ln3+ binding domains I and II in calmodulin. Eu3+ luminescence experiments with Zn2+-loaded S100 beta show that the lifetime for Eu3+ in site I in Zn2+-loaded S100 beta is significantly different than that in the absence of Zn2+. Tyrosine-17-sensitized Tb3+ luminescence experiments indicate that the Tb3+ occupying the proximal weaker binding site I is sensitized, whereas Tb3+ in site II is not. The distance between sites I and II (15.0 +/- 0.4 A) in S100 beta was determined from Forster-type energy transfer in D2O solutions containing bound Eu3+ donor and Nd3+ acceptor ions. For Zn2+-S100 beta, the intersite distance is reduced to 13 +/- 0.3 A. Location of histidine-15 close to pseudo-EF site I suggests that Zn2+ binding likely changes the conformation of this site, causing a reduction of the intersite distance by approximately 2 A.     

Core mutations that promote the calcium-induced allosteric transition of bovine recoverin

Recoverin is a small calcium binding protein involved in regulation of the phototransduction cascade in retinal rod cells. It functions as a calcium sensor by undergoing a cooperative, ligand-dependent conformational change, resulting in the extrusion of the N-terminal myristoyl group from a hydrophobic pocket. To test the role of certain core residues in tuning this allosteric switch, we have made and characterized two mutants: W31K, which replaces Trp31 with Lys; and a double mutant, I52A/Y53A, in which Ile52 and Tyr53 are both replaced by Ala. These mutations decrease the hydrophobicity of the myristoyl binding pocket. They are thus expected to make sequestering of the myristoyl group less favorable and destabilize the Ca2+-free state. As predicted, the myristoylated forms of the mutants exhibit increased affinity for Ca2+, whether monitored by equilibrium binding of 45Ca2+ (Kd = 17.2, 7.9, and 8.1 microM for wild type, W31K, and I52A/Y53A, respectively) or by the change in tryptophan fluorescence associated with the conformational change (Kd = 17.9, 3.6, and 4.4 microM for wild type, W31K, and I52A/Y53A, respectively). The mutants also exhibit decreased cooperativity of binding (Hill coefficient = 1.2 and 1.0 for W31K and I52A/Y53A vs 1. 4 for wild type). Binding of the mutant proteins to rod outer segment membranes occurs at lower Ca2+ concentrations compared to wild-type protein (K1/2 = 5.6, 2.2, and 1.0 microM for wild type, W31K, and I52A/Y53A, respectively). The unmyristoylated forms of the mutants exhibit biphasic Ca2+ binding curves, nearly identical to that observed for wild type. The binding data for the two mutants can be explained by a concerted allosteric model in which the mutations affect only the equilibrium constant L between the two allosteric forms, T (the Ca2+-free form) and R (the Ca2+-bound form), without affecting the intrinsic binding constants for the two Ca2+ sites. Two-dimensional NMR spectra of the Ca2+-free forms of the mutants have been compared to the wild-type spectrum, whose peaks have been assigned to specific residues (1). Many resonances assigned to residues in the C-terminal domain (residues 100-202) in the wild-type spectrum are identical in the mutant spectra, suggesting that the backbone structure of the C-terminal domain is probably unchanged in both mutants. The N-terminal domain, in which both mutations are located, reveals in each case numerous changes of undetermined spatial extent.     

Tryptophan exposure in various conformational isomers of bovine prothrombin fragment 1. An acrylamide quenching study

In order to assess the importance of a variety of environmental factors on the structure of bovine prothrombin fragment 1, we have examined acrylamide quenching of fragment 1 intrinsic fluorescence. Tryptophan exposure, determined from Stern-Volmer plots, is heterogeneous with one or more of the three fragment 1 tryptophans being exposed to solvent. In the presence of Ca2+ or Mg2+ even the most accessible tryptophan(s) are relatively buried. Only small differences in tryptophan exposure may exist between fragment 1-Ca2+ and fragment 1-Mg2+ complexes. Lowering pH, on the other hand, results in increased tryptophan exposure. Finally, structural isomers of fragment 1 which exist in the absence of metal ions have identical tryptophan exposure as determined by acrylamide quenching and fluorescence intensity.     

Study on Anticoagulation Factor I from Agkistrodon Acutus Venom by Rare Earth Ion Probes

Ｓｎａｋｅｖｅｎｏｍｓｃｏｎｔａｉｎｃｏｍｐｌｅｘｐｒｏｔｅｉｎｓｗｈｉｃｈｐｏｓｓｅｓｓｖａｒｉｏｕｓｂｉｏｌｏｇｉｃａｌａｃｔｉｖｉｔｉｅｓ ,ｉｎ ｃｌｕｄｉｎｇａｃｌａｓｓｔｈａｔａｆｆｅｃｔｓｃｏａｇｕｌａｔｉｏｎｓｙｓｔｅｍｗｉｔｈｃｏａｇｕｌａｎｔｏｒａｎｔｉｃｏａｇｕｌａｎｔａｃｔｉｖｉｔｉｅｓ .Ａｎａｎｔｉｃｏａｇｕｌａｎｔｆａｃｔｏｒ (ＡＣＦ)ｗａｓｐｒｅｖｉｏｕｓｌｙｉｓｏ ｌａｔｅｄｆｒｏｍｔｈｅｖｅｎｏｍｏｆＡｇｋｉｓｔｒｏｄｏｎａｃｕｔｕｓｆｒｏｍｓｏｕｔｈｅｒｎＡｎｈｕ…     

An anti-inflammatory role for glucocorticoids in the ovaries?

The binding of Ca2+ ions to bovine and human thyroglobulin (Tg) was demonstrated qualitatively by 45Ca overlay on polyvinylidene difluoride (PVDF) membranes. A quantitative analysis of the interaction of metal ions with bovine Tg was conducted by fluorimetric titration of the protein with Tb3+ ions. These have been used with several proteins as isomorphous replacement probes for Ca2+ ions, as protein-bound Tb3+ ions fluoresce, upon irradiation in the UV region, because of energy transfer from tyrosyl and/or tryptophanyl residues. The fluorescence emission spectrum of Tg excited at 280 nm showed, upon addition of Tb3+ ions, a peak at 546 nm and a marked decrease at 335 nm, indicating an efficient F?rster energy transfer between bound Tb3+ ions and closely located Tg intrinsic chromophores. Titration of Tg with Tb3+ ions, carried out by monitoring the emitted fluorescence at 546 nm, indicated the presence of 13.15 metal binding sites per Tg molecule.     

Solution structure of calcium-bound rat S100B(betabeta) as determined by nuclear magnetic resonance spectroscopy,

The three-dimensional structure of Ca2+-bound rat S100B(betabeta) has been determined using data from a series of two-dimensional (2D), three-dimensional (3D), and four-dimensional (4D) nuclear magnetic resonance (NMR) experiments. Each S100beta subunit (91 residues) contains four helixes (helix 1, E2-R20; helix 2, K29-N38; helix 3, Q50-D61; and helix 4, F70-A83) and one antiparallel beta-sheet (strand 1, K26-K28; and strand 2, E67-D69) which brings the normal and pseudo EF-hands together. As found previously for rat apo-S100B(betabeta) [Drohat, A. C., et al. (1996) Biochemistry 35, 11577-11588], helixes 1, 1', 4, and 4' associate to form an X-type four-helix bundle at the symmetric dimer interface. Additionally, Ca2+ binding does not significantly change the interhelical angle of helixes 1 and 2 in the pseudo EF-hand (apo, Omega1-2 = 132 +/- 4 degrees; and Ca2+-bound, Omega1-2 = 137 +/- 5 degrees). However, the interhelical angle of helixes 3 and 4 in the normal EF-hand (Omega3-4 = 106 +/- 4 degrees) changed significantly upon the addition of Ca2+ (DeltaOmega3-4 = 112 +/- 5 degrees) and is similar to that of the Ca2+-bound EF-hands in calbindin D9K, calmodulin, and troponin (84 degrees 相似文献   

Early destruction of tryptophan residues of apolipoprotein B is a vitamin E-independent process during copper-mediated oxidation of LDL

The decrease of the tryptophan fluorescence (Ex/Em = 282/331 nm) was used to monitor the kinetics of copper-mediated LDL oxidation. Cu2+ causes a concentration-dependent quenching of the LDL Trp-fluorescence, the maximum of about 22% suggests that 8-9 Trp residues (out of a total of 37) are accessible for Cu2+ ions. Decomposition of LDL tryptophan commences immediately after addition of Cu2+ and proceeds in two stages with quite different rates. At a molar ratio of Cu2+/LDL = 33:1 the LDL particle looses 1 Trp every 13.5 min in the initial slow phase and every 4.1 min in the subsequent rapid The second, stage temporarily coincides with the propagating lipid peroxidation. In the initial phase loss of Trp proceeds with a constant rate for up to 200 min depending on the copper concentration. Whereas lipid peroxidation accelerates after consumption of vitamin E, rate of Trp loss does not increase. Loading of LDL with vitamin E has also no effect on the initial rate of Trp loss. During the initial phase a loss of one Trp residue/LDL is accompanied by the loss of two alpha-tocopherols and the generation of two conjugated lipid hydroperoxides. The results suggest Trp residues play a role in initiating the lipid peroxidation process in the LDL particle. In such kinetic studies, precautions must be taken to avoid photodecomposition of LDL-Trp. The LDL vitamin E fluorescence (Ex/Em = 290/323 nm) does not interfere with the Trp fluorescence even at high concentrations.     

The pseudo-beta I-turn. A new structural motif with a cis peptide bond in cyclic hexapeptides

Synthesis and conformational analysis of three cyclic hexapeptides cyclo(-Gly1-Pro2-Phe3-Val4-Xaa5-Phe6), Xaa = Phe (I), D-Phe (II) and D-Pro (III), were carried out to examine the influence of proline on the formation of reverse turns and the dynamics of hydrophobic peptide regions. Assignment of all 1H and 13C resonances was achieved by homo- and heteronuclear 2D-NMR techniques (TOCSY, ROESY, HMQC, HMQC-TOCSY and HMBCS-270). The conformational analysis is based on interproton distances derived from ROESY spectra and homo- and heteronuclear coupling constants (E.COSY, HETLOC and HMBCS-270). For structural refinements restrained molecular dynamics (MD) simulations in vacuo and in DMSO were performed. Each peptide exhibits two conformations in DMSO solution due to cis-trans isomerism about the Gly-Pro peptide bond. Surprisingly the cis-Gly-Pro segment in the minor isomers is not involved in a beta VI-turn, but forms a turn structure with cis-Gly-Pro in the i and i + 1 positions. Although no stabilizing hydrogen bond is found in this turn, the phi- and psi-angles closely correspond to a beta I-turn [Pro2: phi(i + 1) -60 degrees, psi(i + 1) -30 degrees; Phe3: phi(i + 2) -100 degrees, psi(i + 2) -50 degrees]. Hence we call this structural element a pseudo-beta I-turn. As expected, in the dominating all-trans isomers proline occupies the i + 1 position of a standard beta I-turn. Therefore, cis-trans isomerization of the Gly1-Pro2 amide bond only induces a local conformational rearrangement, with minor structural changes in other parts of the molecule. However, the geometry of the other regions is affected by the chirality of the i + 1 amino acid for both isomers (beta I for Phe5, beta II' for D-Phe5 or D-Pro5).     

Purification and characterization of a Ca(2+)-binding 450-kDa protein (MCBP-450) in the plasma membrane-enriched fraction from a molluscan smooth muscle

In accordance with physiological and electronmicroscopic evidence that, in the anterior byssal retractor muscle (ABRM) of a common mussel Mytilus edulis, Ca2+ activating the contractile system is accumulated at the inner surface of the plasma membrane and at the membrane of sarcoplasmic reticulum (Ebashi, S. and Endo, M. (1968) Prog. Biophys. Mol. Biol. 18., 123-183; Suzuki, S. and Sugi, H. (1982) in The role of calcium in biological systems, Vol. I (Anghileri, L.J. and Tuffet-Anghileri, A.M., eds.), pp. 201-207, CRC Press, Boca Raton), we have found a high-molecular-mass (450 kDa) Ca(2+)-binding protein (MCBP-450) in the membrane fractions of the ABRM by 45Ca autoradiography of proteins transferred to nitrocellulose membrane (Rüegg, J. C. (1971) Physiol. Rev. 51, 201-248). MCBP-450, purified to electrophoretic homogeneity, exhibited Ca(2+)-dependent changes in mobility, tryptophan fluorescence, UV absorption and CD spectrum, indicating its Ca(2+)-dependent conformational changes. MCBP-450 has a high content of aspartic and glutamic acid (23.8%) and a high content of basic residues (27%). It has a high capacity Ca(2+)-binding site, which binds about 38 mol of Ca2+ per mol with an adissociation constant of 10(4) M-1, and a low-capacity Ca(2+)-binding site, which binds about 7 mol of Ca2+ per mol with an association constant of 10(5) M-1. These characteristics of MCBP-450 are consistent with the view that it is actually involved in regulating the contraction-relaxation cycle in the ABRM.     

ATP and acetyl phosphate induces molecular events near the ATP binding site and the membrane domain of Na+,K+-ATPase. The tetrameric nature of the enzyme

The addition of ATP to Mg2+-Na+-bound-probe labeled Na+,K+-ATPase preparations containing approximately 0.5 mol of pyridoxal 5'-diphospho-5'-adenosine (AP2PL) probe at Lys-480 and approximately 0.9 mol of fluorescein 5'-isothiocyanate (FITC) probe at Lys-501 showed a decrease and an increase in the AP2PL fluorescence intensity with neither significant ATP-dependent phosphorylation nor FITC fluorescence change. The rate constants for the fluorescence change increased nearly linearly with increasing ATP concentrations. The substitution of AcP for ATP decreased the FITC fluorescence rather monophasically, 8.5/s, which was followed by the half-site phosphorylation with same amount of components with different rate constant, 7.2 and 4.6/s, followed by a much slower increase in the two components of AP2PL fluorescence, 1.4 and 0.2/s. The addition of Na+ with increasing concentrations of ATP to the K+-bound AP2PL-FITC enzymes induced accelerations in the decrease and an increase in the AP2PL fluorescence intensity with two different increases in the FITC fluorescence intensity, showing that the same concentration of ATP is capable of inducing four different fluorescence changes. The addition of ATP to the Mg2+-Na+-bound enzymes modified with N-[p-(2-benzimidazolyl)phenyl]-maleimide (BIPM) at Cys-964 and retaining full Na+,K+-ATPase activity induced two different increases in BIPM fluorescence intensity. Each rate constant for the BIPM fluorescence change versus concentrations of ATP gave two intersecting straight lines. These data and the stoichiometries of fluorescence probe bindings and ATP- and AcP-dependent phosphorylation provide strong support for the conclusion that the functional membrane-bound Na+,K+-ATPase is a tetramer.     

Ca2+ activation of wheat peroxidase: a possible physiological mechanism of control

Peroxidation of substrates such as ascorbic acid, pyrogallol, or ferulic acid, as well as indole acetic acid oxidation catalyzed by wheat germ peroxidase (WGP)2 C2, were found to be activated by Ca2+. This activation is independent of the stabilizing effect of structural Ca2+ reported for peroxidases. Steady state kinetics of ferulic acid oxidation catalyzed by WGP C2 showed an increase in the rate of compound I formation and of compound II decomposition in the presence of the ion, evidenced as an increase in rate constants k1, from 8.9 x 10(5) to 4.5 x 10(5) M-1 cm-1, and k3, from 4.4 x 10(5) to 1.1 x 10(6) M-1 cm-1. The dissociation constant Kd, for the cyanide derivative of the enzyme showed a marked decrease from 220 to 34 microM in the presence of Ca2+, thus implying an effect of the ion in the H2O2 binding step. In the presence of Ca2+, a conformational change in the protein was revealed by tryptophan fluorescence, providing a basis for the activation mechanism. Other peroxidases such as horseradish peroxidase and WGP C3 were not activated by Ca2+. The results suggest the existence of a physiological mechanism of control of peroxidase isozymes activity mediated by Ca2+.     

Unfolding kinetics of bovine trypsinogen

The unfolding kinetics of bovine trypsinogen were studied by a fluorescence-detected stopped-flow technique at pH 5.8. Trypsinogen unfolding appeared to be a rather complex reaction. Two phases, fast (with a time constant in the millisecond range) and slow, were detected in the range 2-7 M guanidium chloride (GdmCl). The natural logarithm of the rate constant of the slow phase exhibited strong dependence on [GdmCl], changing from hundreds of seconds at low denaturant concentration to about 20 ms at 7 M GdmCl. The curvature of this dependence further suggests a complex mechanism of unfolding. Generally, similar kinetics were observed for the trypsinogen.Ca complex. Small differences could be noticed, however, for the fast phase. In agreement, Ca2+ influenced only this stage of the reaction. Analysis of the dependence of the time constant of the fast phase on [CaCl2] indicates that at 4 M GdmCl, trypsinogen.Ca unfolds about sixfold slower than free zymogen, and that native trypsinogen at 4 M GdmCl still exhibits high affinity for Ca2+. Limited data on trypsin unfolding show virtually an identical dependence of the slow phase on [GdmCl]; the fast phase, however was not observed. Moreover, in the 3-4.5 M GdmCl range, a separate phase was detected. It is postulated that this phase is a manifestation of the activation-domain unfolding. The Eyring plots for the fast phase of . trypsinogen and trypsinogen.Ca unfolding are linear, indicating little change in heat capacity for this stage of reaction. The slow step of unfolding, however, shows significant curvature which indicates a substantial increase in heat capacity.     

Use of retinoic acid as a fluorescent probe to study conformational change of the albumin molecule

The binding of retinoic acid to serum albumin induces quenching of the protein fluorescence when it is excited at 280 nm, on the other hand the bound ligand acquires intrinsic fluorescence. Albumin has two kinds of binding sites for retinoic acid with an affinity constant of 10(5) M-1 and 10(4) M-1 respectively. The binding is entropically driven and produces a conformational change at the environment of the albumin tryptophan residues. This change was described by an equilibrium constant assuming two conformational states of the albumin tryptophan residues. Retinoic acid binds to the albumin fatty acid binding sites, producing a perturbation in the warfarin and benzodiazepine binding sites of this protein.     

Structural changes of sarcoplasmic reticulum Ca(2+)-ATPase upon Ca2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence

Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca2+ from the photolabile Ca2+ chelator 1-(2-nitro-4,5-dimethoxy)-N,N,N',N',- tetrakis[(oxycarbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H2O and 2H2O were detected in the spectral region from 1800 cm-1 to 1200 cm-1, reflecting photolysis of DM-nitrophen and Ca2+ binding to the Ca(2+)-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca2+ binding activity of the ATPase in this specific step. Ca2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm-1 can be assigned to Ca2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm-1 to 6 cm-1 upon 1H2O/2H2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262-6267).     

Comparison of the Ca2+-binding properties of human recombinant calretinin-22k and calretinin

Calretinin-22k (CR-22k) is a splice product of calretinin (CR) found specifically in cancer cells, and possesses four EF-hands and a differently processed C-terminal end. The Ca2+-binding properties of recombinant human calretinin CR-22k were investigated by flow dialysis and spectroscopic methods and compared with those of CR. CR possesses four Ca2+-binding sites with positive cooperativity (nH = 1.3) and a [Ca2+]0.5 of 1.5 microM, plus one low affinity site with an intrinsic dissociation constant (K'D) of 0.5 mM. CR-22k contains three Ca2+-binding sites with nH of 1.3 and [Ca2+]0.5 of 1.2 microM, plus a low affinity site with K'D of 1 mM. All the sites seem to be of the Ca2+-specific type. Limited proteolysis and thiol reactivity suggest that that the C terminus of full-length CR, but not of CR-22k, is in close proximity of site I leading to mutual shielding. Circular dichroism (CD) spectra predict that the content of alpha-helix in CR and CR-22k is similar and that Ca2+ binding leads to very small changes in the CD spectra of both proteins. The optical properties are very similar for CR-22k and CR, even though CR-22k possesses one additional Trp at the C-terminal end, and revealed that the Trp residues are organized into a hydrophobic core in the metal-free proteins and become even better shielded from the aqueous environment upon binding of Ca2+. The fluorescence of the hydrophobic probe 2-p-toluidinylnaphtalene-6-sulfonate is markedly enhanced by the two proteins already in the absence of Ca2+ and is further increased by binding of Ca2+. The trypsinolysis patterns of CR and CR-22k are markedly dependent on the presence or absence of Ca2+. Together, our data suggest the presence of an allosteric conformational unit encompassing sites I-III for CR-22k and I-IV for CR, with a very similar conformation and conformational changes for both proteins. In the allosteric unit of CR, site IV is fully active, whereas in CR-22k this site has a 80-fold decreased affinity, due to the decreased amphiphilic properties of the C-terminal helix of this site. Some very specific Ca2+-dependent conformational changes suggest that both CR and CR-22k belong to the "sensor"-type family of Ca2+-binding proteins.