A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies

Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.     

Structural determinants of Ca2+ exchange and affinity in the C terminal of cardiac troponin C

The C terminal of cardiac troponin C (TnC) has two Ca2+-Mg2+ sites which exhibit approximately 20-fold higher Ca2+ affinity than the two C-terminal Ca2+ specific sites in calmodulin (CaM). Substitution of the third EF-hand of TnC for the corresponding EF-hand of CaM produced a mutant (CaM[3TnC]) with a 10-fold higher C-terminal Ca2+ and Mg2+ affinity. Substitution of loop 3 of TnC for loop 3 of CaM produced a mutant (CaM[loop3TnC]) with a 10-fold faster Ca2+ on rate and a 5-fold faster Ca2+ off rate than CaM. A mutant CaM (CaM[loop3X, Z]) which contained the identical coordinating amino acids and X and Z acid pairs of TnC loop 3 had a 3-fold higher C-terminal Ca2+ affinity without the increased Ca2+ exchange rates exhibited by CaM[loop3TnC]. Thus, loop factors other than the acid pairs must be responsible for the rapid Ca2+ exchange rates of CaM[loop3TnC]. Helix 6 and helix 5 in the third EF-hand of TnC support the rapid Ca2+ on rate of TnC's loop 3 and produce an approximately 4-fold reduction in its Ca2+ off rate, explaining the high Ca2+ affinity of the third EF-hand of TnC. Exchanging loop 3 or helix 5 of TnC into CaM increased the Mg2+ affinity by decreasing the Mg2+ off rate. Our results are consistent with the high Ca2+ and Mg2+ affinity of the third EF-hand of TnC resulting from the two (X and Z) acid pairs in loop 3, coupled with the greater hydrophobicity of helix 6 and helix 5 compared to that of the third EF-hand of CaM.     

The current and future outlook in immunosuppressive therapy for children

An alignment of amino acid sequences suggests that the spectrin domain, which contains two EF-hand calcium-binding motifs, is structurally related to calmodulin. It is possible to align approximately 160 residues at the C-terminus of alpha-spectrin with the entire calmodulin sequence. We have expressed this domain in Escherichia coli and purified it. Circular dichroic and nuclear magnetic resonance spectroscopy show that the protein is folded and mostly helical. The conformation of the protein, as monitored spectroscopically, is sensitive to calcium at 0.1-1.0 mM. Equilibrium dialysis shows that there are two binding sites within this domain, with affinities in the 0.5 mM range. The domain can be split into N-terminal and C-terminal halves which fold independently. Only the N-terminal subdomain binds calcium. These data suggest that the C-terminus of alpha-spectrin has a domain with a calmodulin fold and two calcium-binding sites. Sequence alignments suggest that the related domains in alpha-actinin, and possibly in dystrophin, may share the same calmodulin-like structure. However, only non-muscle alpha-actinins appear to have one or two EF-hand(s) with the calcium-binding consensus sequence, and a strict consensus is not found in the muscle alpha-actinins or dystrophins.     

Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana

Calcium-dependent protein kinases(CDPKs)comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule.So far,a biological function in abiotic stress signaling has only been reported for few CDPK isoforms,whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown.Here,we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress.Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation.In transgenic Arabidopsis complementation lines in the cpk21 mutant background,in which either CPK21 wildtype,or a full-length enzyme variant carrying an amino-acid substitution were stably expressed,stress responsitivity was restored by CPK21 but not with the kinase inactive variant.The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain,N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity,suggesting a crucial role for the N-terminal EF-hand pair.Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.     

Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40

BM-40 (also known as SPARC or osteonectin) is an anti-adhesive secreted glycoprotein involved in tissue remodelling. Apart from an acidic N-terminal segment, BM-40 consists of a follistatin-like (FS) domain and an EF-hand calcium-binding (EC) domain. Here we report the crystal structure at 3.1 A resolution of the FS-EC domain pair of human BM-40. The two distinct domains interact through a small interface that involves the EF-hand pair of the EC domain. Residues implicated in cell binding, inhibition of cell spreading and disassembly of focal adhesions cluster on one face of BM-40, opposite the binding epitope for collagens and the N-linked carbohydrate. The elongated FS domain is structurally related to serine protease inhibitors of the Kazal family. Notable differences are an insertion into the inhibitory loop in BM-40 and a protruding N-terminal beta-hairpin with striking similarities to epidermal growth factor. This hairpin is likely to act as a rigid spacer in proteins containing tandemly repeated FS domains, such as follistatin and agrin, and forms the heparin-binding site in follistatin.     

Hydrophobic core substitutions in calbindin D9k: effects on stability and structure

The effects of hydrophobic core mutations on the stability and structure of the four-helix calcium-binding protein, calbindin D9k, have been investigated. Eleven mutations involving eight residues distributed within the hydrophobic core of calbindin D9k were examined. Stabilities were measured by denaturant and thermal induced unfolding monitored by circular dichroism spectroscopy. The mutations were found to exert large effects on the stability with midpoints in the urea induced unfolding varying from 1.8 M for Leu23 --> Gly up to 6.6 M for Val70 --> Leu and free energies of unfolding in the absence of denaturant ranging from 6.6 to 27.4 kJ/mol for the Phe66 --> Ala mutant and the wild-type, respectively. A significant correlation was found between the difference in free energy of unfolding (Delta Delta GNU) and the change in the surface area of the side chain caused by the mutation, in agreement with other studies. Notably, both increases and decreases in side-chain surface area caused quantitatively equivalent effects on the stability. In other words, a correlation between the absolute value of the change in the surface of the side chain and Delta DeltaGNU was observed with a value of approximately 0.14 kJ M-1 A-2. The generality of this observation is discussed. Significant effects on the cooperativity of the unfolding reaction were also observed. However, a correlation between the cooperativity and Delta Delta GNU, which has been reported in other systems as an indication of effects of mutations on the unfolded state, was not observed for calbindin D9k. Despite the large effects on Delta Delta GNU and cooperativity, the structures of the mutants in the native form remained intact as indicated by circular dichroism, NMR, and fluorescence measurements. The structural response to calcium-binding was also conserved. The following paper in this issue [Kragelund, B. B., et al. (1998) Biochemistry 37, 8926-8937] examines the effects of these mutations on the calcium binding properties of calbindin D9k.     

A change-in-hand mechanism for S100 signalling

S100 proteins are a group of small dimeric calcium-binding proteins making up a large subclass of the EF-hand family of calcium-binding proteins. Members of this family of proteins have been proposed to act as intracellular calcium modulatory proteins in a fashion analogous to that of the EF-hand sensor proteins troponin-C and calmodulin. Recently, NMR spectroscopy has provided the three-dimensional structures of the S100 family members S100A6 and S100B in both the apo- and calcium-bound forms. These structures have allowed for the identification of a novel calcium-induced conformational change termed the change-in-hand mechanism. Helix III of the C-terminal calcium-binding loop changes its helix-helix interactions (or handness) with the remainder of the molecule primarily owing to the reorientation of the backbone in an effort to coordinate the calcium ion. This reorientation of helix III exposes several residues in the C-terminus and linker regions of S100B resulting in the formation of a hydrophobic patch surrounded be a number of acidic residues. This site is the proposed region for protein-protein recognition.     

A protein folding intermediate of ribonuclease T1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy

The rate-limiting step during the refolding of S54G/P55N ribonuclease T1 is determined by the slow trans-->cis prolyl isomerisation of Pro39. We investigated the refolding of this variant by one-dimensional (1D) and two-dimensional (2D) real-time NMR spectroscopy, initiated by a tenfold dilution from 6 M guanidine hydrochloride at 10 degreesC. Two intermediates could be resolved with the 1D approach. The minor intermediate, which is only present early during refolding, is largely unfolded. The major intermediate, with an incorrect trans Pro39 peptide bond, is highly structured with 33 amide protons showing native chemical shifts and native NOE patterns. They could be assigned in a real-time 2D-NOESY (nuclear Overhauser enhancement spectroscopy) by using a new assignment strategy to generate positive and negative signal intensities for native and non-native NOE cross-peaks, respectively. Surprisingly, amide protons with non-native environments are located not only close to Tyr38-Pro39, but are spread throughout the entire protein, including the C-terminal part of the alpha-helix, beta-strands 3 and 4 and several loop regions. Native secondary and tertiary structure was found for the major intermediate in the N-terminal beta-strands 1 and 2 and the C terminus (connected by the disulfide bonds), the N-terminal part of the alpha-helix, and the loops between beta-strands 4/5 and 5/6. Implications of these native and non-native structure elements of the intermediate for the refolding of S54G/P55N ribonuclease T1 and for cis/trans isomerizations are discussed.     

Determination of calcium-binding sites in rat brain calbindin D28K by electrospray ionization mass spectrometry

Calbindin D28K, a member of the troponin-C superfamily of calcium-binding proteins, contains six putative EF-hand domains. Calcium-binding studies of the protein by different groups of investigators have yielded discordant results with respect to the stoichiometry of calcium-binding. It has been suggested that the protein binds anywhere from 3-6 mol of calcium/mol of protein. We used negative ion electrospray ionization mass spectrometry in order to definitively determine the exact calcium-binding stoichiometry of calbindin D28K and two mutant forms of the protein, one lacking EF-hand 2 (delta2) and the other lacking EF-hands 2 and 6 (delta2,6). The full-length protein bound 4 mol of calcium/mol of protein, while both of the deletion mutants bound 3 mol of calcium. Since terbium has been used extensively as a probe for the determination of the calcium-binding stoichiometries of calcium-binding proteins, we also examined the binding of terbium to the three proteins under the same conditions. Full-length calbindin D28K bound 4 mol of terbium/mol of protein, while calbindin delta2 and delta2,6 each bound 3 mol. These results clearly show that calbindin D28K binds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.     

Cation binding and conformation of tryptic fragments of Nereis sarcoplasmic calcium-binding protein: calcium-induced homo- and heterodimerization

Nereis sarcoplasmic calcium-binding protein (NSCP) is a compact 20-kDa protein that competitively binds three Ca2+ or Mg2+ ions and displays strong positive cooperativity. Its three-dimensional structure is known. It thus constitutes a good model for the study of intramolecular information transduction. Here we probed its domain structure and interaction between domains using fragments obtained by controlled proteolysis. The metal-free form, but not the Ca2+ or Mg2+ form, is sensitive to trypsin proteolysis and is preferentially cleaved at two peptide bonds in the middle of the protein. The N-terminal fragment 1-80 (T1-80) and the C-terminal fragment 90-174 (T90-174) were purified to electrophoretic homogeneity. T1-80, which consists of a paired EF-hand domain, binds one Ca2+ with Ka = 3.1 x 10(5) M-1; entropy increase is the main driving force of complex formation. Circular dichroism indicates that T1-80 is rich in secondary structure, irrespective of the Ca2+ saturation. Ca2+ binding provokes a difference spectrum which is similar to that observed in the intact protein. These data suggest that this N-terminal domain constitutes the stable structural nucleus in NSCP to which the first Ca2+ binds. T90-174 binds two Ca2+ ions with Ka = 3.2 x 10(4) M-1; the enthalpy change contributes predominantly to the binding process. Metal-free T90-174 is mostly in random coil but converts to an alpha-helical-rich conformation upon Ca2+ binding. Ca2+ binding to T1-80 provokes a red-shift and intensity decrease of the Trp fluorescence but a blue-shift and intensity increase in T90-174.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger

The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+ gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a "P-loop"-like structure.     

The neurosteroid pregnenolone sulfate infused into the nucleus basalis increases both acetylcholine release in the frontal cortex or amygdala and spatial memory

GTP:AMP phosphotransferase (adenylate kinase isozyme 3, AK3) mutants were obtained by using the ability of AK3 to complement a temperature-sensitive mutation of Escherichia coli adenylate kinase (AKe). Five mutants, P16L, G19S, G91D, G91S, and P93L, had mutation sites located at two loops that are involved in substrate binding of the enzyme. P16L and G19S bearing changes at the first loop showed reduced affinity for both GTP and AMP, the extent of reduction being slightly higher for GTP than AMP. In contrast, G91S and P93L having alterations at the second loop had lower affinities for AMP. Only the alterations at the second loop strongly influenced the Vmax value of the enzyme. Another mutant, D163N, had a substitution at the site forming a salt bridge in adenylate kinase isozyme 1 (AK1), which influenced the Vmax as well as the Km values for both substrates. The kinetic characteristics of these mutants were comparable to those of the corresponding AK1 or AKe mutants. Furthermore, from the results of mutations T201P and T201A that had alterations in all the kinetic parameters of AK3 and from a comparison with the structure and the kinetic parameters of AKe, we expect that a residue(s) around Thr201 is involved in recognition of the base of nucleoside triphosphate.     

Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual-site Ca2+ coordination

Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.     

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.     

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Escherichia coli DNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43 kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, compete in vivo with the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoiling in vivo. This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activity in vivo. Thus, our in vivo approach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or GyrB/GyrB, or GyrB/GyrA protein interactions.     

The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions

The Src protein tyrosine kinase plays a critical role in a variety of signal transduction pathways. Strict regulation of its activity is necessary for proper signalling. We present here the crystal structure of chicken Src which is phosphorylated at Tyr527 and represents its least active form. Our structure, similar to the recently reported human Hck and Src structures, contains the SH3, SH2 and the kinase domains and the C-terminal regulatory tail but not the N-terminal unique domain. The SH3 domain uses its hydrophobic surface to coordinate the SH2-kinase linker such that residues Gln251 and Leu255 specifically interact with side chains in the beta2-beta3 and the alphaC-beta4 loops of the N-terminal lobe opposite of the kinase active site. This position of the SH3 domain and the coordination of the SH2-kinase linker also optimally places the SH2 domain such that the phosphorylated Tyr527 in the C-terminal tail interacts with the SH2 binding pocket. Analogous to Cdk2 kinase, the position of the Src alphaC-helix in the N-terminal lobe is swung out disrupting the position of the active site residues. Superposition of other protein kinases including human Hck and Src onto chicken Src indicate that the alphaC-helix position is affected by the relative position of the N-terminal lobe with respect to the C-terminal lobe of the kinase and that the presence of the SH3/SH2-kinase linker/N-terminal lobe interactions restricts the kinase lobes and alphaC-helix access to the active conformation. These superpositions also suggest that the highly conserved alphaC-beta4 loop restricts the conformational freedom of the N-terminal lobe by anchoring it to the C-terminal lobe. Finally, based on sequence alignments and conservation of hydrophobic residues in the Src SH2-kinase linker as well as in the alphaC-beta4 and beta2-beta3 loops, we propose that the Src-related kinases, Abl, Btk and Csk, share the same quaternary structure.     

Introduction of a proline residue into position 31 of the loop of the dimeric 4-alpha-helical protein ROP causes a drastic destabilization

The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-alpha-helical-bundle protein ROP causes a reduction in the alpha-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters. The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, delta G0D (buffer, 25 degrees C), has been determined to be 43 kJ (mol dimer)-1. The corresponding ROPwt value is 72 kJ (mol dimer)-1 (Steif et al., 1993). The extrapolated delta G0D values obtained from urea and GdmHCI un- and refolding studies are identical within error limits. Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters. At T1/2 = 43 degrees C (Cprotein = 0.05 mg.ml-1) the ROP A31P mutant is characterized by delta Hv.H.0 = 272 kJ (mol dimer)-1, delta Cp = 7.2 kJ (mol dimer)-1 K-1, delta S0 = 762 J (mol dimer)-1 K-1. These parameters are only approximately 50% as large as the corresponding values of ROPwt. We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure. This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm. All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability.     

Structure of the interhelical loops and carboxyl terminus of bacteriorhodopsin by X-ray diffraction using site-directed heavy-atom labeling

The positions of single amino acids in the interhelical loop regions and the C-terminal tail of bacteriorhodopsin (bR) were investigated by X-ray diffraction using site-directed heavy-atom labeling. Since wild-type bR does not contain any cysteines, appropriate cysteine mutants were produced with a unique sulfhydryl group at specific positions. These sites were then labeled with mercury using the sulfhydryl specific reagent p-chloromercuribenzoate (p-CMB). The cysteine mutants D96A/V101C, V130C, A160C, and G231C were derivatized with labeling stoichiometries of 0.93 +/- 5%, 0.85 +/- 5%, 0.79 +/- 7%, and 0.77 +/- 8%, respectively (Hg per bR). No incorporation was observed with wild-type bR under the same conditions. All mutants and heavy-atom derivatives were fully active as judged by the kinetics of the photocycle and of the proton release and uptake. Moreover, the unit cell dimensions of the two-dimensional P3 lattice were unchanged by the mutations and the derivatization. This allowed the position of the mercury atoms, projected onto the plane of the membrane, to be calculated from the intensity differences in the X-ray diffraction pattern between labeled and unlabeled samples using Fourier difference methods. The X-ray diffraction data were collected at room temperature from oriented purple membrane films at 100% relative humidity without the use of dehydrating solvents. These native conditions of temperature, humidity, and solvent are expected to preserve the structure of the surface-exposed loops. Sharp maxima corresponding to a single mercury atom were found in the difference density maps for D96A/V101C and V130C. Residues 101 and 130 are in the short loops connecting helices C/D and D/E, respectively. No localized difference density was found for A160C and G231C. Residue 160 is in the longer loop connecting helices E and F, whereas residue 231 is in the C-terminal tail. Residues 160 and 231 are apparently in a more disordered and mobile part of the structure.     

Oligomerization and divalent ion binding properties of the S100P protein: a Ca2+/Mg2+-switch model

S100P is a 95 amino acid residue protein which belongs to the S100 family of proteins containing two putative EF-hand Ca2+-binding motifs. In order to characterize conformational properties of S100P in the presence and absence of divalent cations (Ca2+, Mg2+ and Zn2+) in solution, we have analyzed hydrodynamic and spectroscopic characteristics of wild-type and several variants (Y18F, Y88F and C85S) of S100P using equilibrium centrifugation, gel-filtration chromatography, circular dichroism and fluorescence spectroscopies. Analysis of the experimental data shows the following. (1) In agreement with the predictions there are two Ca2+-binding sites in the S100P molecule with different affinity; the high affinity binding site has an apparent binding constant of approximately 10(7) M-1 and the low affinity binding site has an apparent binding constant of approximately 10(4) M-1. (2) The high and low affinity Ca2+-binding sites are located in the C and N-terminal parts of the S100P molecule, respectively. (3) These C and N-terminal sites can also bind other divalent ions. The C-terminal site binds Zn2+ (with relatively low affinity approximately 10(3) M-1), but not Mg2+. The N-terminal site binds Mg2+ with the apparent binding constant approximately 10(2) M-1. (4) Binding of Ca2+ to the C-terminal site and binding of Mg2+ to the N-terminal site occur in the physiological concentration range of these ions (micromolar for Ca2+ and millimolar for Mg2+). (5) Oligomerization state of the S100P molecule appears to change upon addition of Ca2+. On the basis of these observations a plausible model for S100P as a Ca2+/Mg2+ switch has been proposed.