NMR and stopped-flow studies of metal ion binding to alpha-lactalbumins.

1H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to alpha-lactalbumins from bovine, goat, and human sources. We have identified two 1H-NMR markers diagnostic of metal ion binding to the high-affinity Ca2+-binding site of bovine alpha-lactalbumin, namely the signals corresponding to the delta-CH3 groups of Met-90, and a leucine, tentatively assigned to Leu-96. A number of metal ions other than Ca2+ bind to this site in either slow (La3+, Lu3+, Y3+, Sr2+, Sc3+) or fast (Cd2+, Ba2+, Pb2+) exchange. From competition experiments using this approach, we have determined an affinity series for metal ion binding at this site, in which lanthanides and Y3+ bind the strongest (Y3+>La3+, Lu3+>Ca2+>Sr2+>Cd2+, Pb2+, Ba2+>Sc3+). Several metal ions do not alter the 1H spectrum of bovine alpha-lactalbumin, retaining the protein in an 'apo-like' state. Evidence is given to support the notion that the paramagnetic divalent metal ions Co2+ and Cu2+ bind to a second distinct site, termed the 'zinc site', and that His-68 is involved in metal ion coordination. Finally, stopped-flow techniques using the indicator Xylenol orange were employed to obtain lanthanide off-rates for bovine, human, and goat alpha-lactalbumins (bovine and goat alpha-LA: k(off)(s-1) approximately 0.2 to 0.01 from La3+ to Lu3+; human alpha-LA: k(off)(s-1) approximately 0.02 to 0.001 from La3+ to Lu3+). In each case, we found that k(off) values decreased by an order of magnitude across the series, meaning that the dissociation constants for these metal ions are relatively constant. Data for the bovine and goat proteins are virtually identical, while the off-rates for human alpha-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca2+ off-rate for the bovine protein (k(off)(s-1) approximately 2 to 5), determined using the fluorescent indicator, BAPTA.

Infrared studies of interaction between metal ions and Ca(2+)-binding proteins. Marker bands for identifying the types of coordination of the side-chain COO- groups to metal ions in pike parvalbumin (pI = 4.10).

Metal-ligand interactions in the Ca(2+)-binding sites of pike parvalbumin (pI = 4.10) have been examined by Fourier-transform infrared spectroscopy. The region of the COO- antisymmetric stretch provides useful information on the types of coordination of the COO- groups to the metal ions in the Mg(2+)-, Mn(2+)-, and Ca(2+)-bound forms. In the spectrum of the Ca(2+)-bound form, two bands are observed at 1,582 and 1,553 cm-1, whereas, in the spectra of the Mg(2+)- and Mn(2+)-bound forms, bands are observed only in the region around 1,582 cm-1 and no band is found in the region around 1,553 cm-1. The 1,553-cm-1 band of the Ca(2+)-bound form reflects the bidentate coordination of the COO- groups of both Glu-62 in the CD site and Glu-101 in the EF site to the Ca2+ ions, which has been made clear by X-ray analysis as a feature of the Ca(2+)-bound form. Absence of such a band in the spectrum of the Mn(2+)-bound form is consistent with the X-ray structure of this form where both of the two COO- groups are unidentate. These unidentate COO- groups of Glu-62 and Glu-101 in the Mn(2+)-bound form seem to give rise to a band at 1,577-1,574 cm-1. The spectrum of the Mg(2+)-bound form is also consistent with the 'pseudo-bridging' coordination of the COO- group of Glu-101 reported in the X-ray structure of a form where the Mg2+ ion occupies only the EF site, and the same spectrum is further indicative of the 'pseudo-bridging' coordination of the COO- group of Glu-62.

Cadmium-113 NMR studies of bovine and human alpha-lactalbumin and equine lysozyme.

The high-affinity calcium-binding sites of bovine and human alpha-lactalbumin as well as equine lysozyme were analyzed by 113Cd NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched 113Cd2+ results in a signal at delta = -75.9 ppm corresponding to the metal ion bound to the lone Ca(2+)-binding site of the protein. A peak at virtually the identical resonance position (delta = -77.1 ppm) was observed in the analogous experiment with bovine alpha-lactalbumin. In addition, a signal upfield of these (delta = -94.7 ppm) was observed for 113Cd(2+)-substituted human alpha-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca(2+)-binding proteins. The field dependence of the 113Cd signals for all three proteins and bovine calmodulin were compared. At each field, the 113Cd signal linewidths for the alpha-lactalbumins and the lysozyme are somewhat broader than those observed for the EF-hand protein. In addition, the 113Cd linewidths for the lactalbumins and the lysozyme, especially bovine alpha-lactalbumin, increase dramatically with the square of the magnetic field strength, indicative of the presence of nuclear relaxation via chemical shift anisotropy and chemical exchange. The protein-bound 113Cd signals for the alpha-lactalbumins are also markedly affected by changes in the amount of K+ present, since Cd2+ and K+ can compete for occupation of the high-affinity Ca(2+)-site. Their linewidths also to some extent depend on the concentration of the protein itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and function of calcium-binding proteins.

The large calcium gradient across the plasma membrane creates different environments for intra- and extracellular calcium-binding proteins. The latter are continuously surrounded by 10(-3) M Ca2+, which promotes activation or stabilization of certain proteases, nucleases, or lipases. Other proteins, such as those involved in blood clotting, contain polyelectrolyte regions that are composed of carboxyglutamic or phosphoserine moieties that allow them to interact with Ca2+. In contrast, intracellular calcium-binding proteins, such as calmodulin and troponin C, the trigger proteins for muscle contractions, need to respond to an increase in Ca2+ from 10(-7) to 10(-6) M during cell activation. Evidence is presented that the pairwise arrangement of characteristic helix-loop-helix calcium-binding sites can result in the positive cooperative binding of Ca2+. This can be further promoted by the binding of ligands, drugs, or target proteins Several drug binding sites on calmodulin are allosterically related and their localization on the unusual dumbbell structure of this molecule will be discussed.

Calcium-43 NMR studies of calcium-binding lysozymes and alpha-lactalbumins.

The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by 43Ca NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective 43Ca NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta approximately -3 to -7 ppm) and quadrupole coupling constants (chi approximately 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pKa values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine alpha-lactalbumin approximately human alpha-lactalbumin greater than pigeon lysozyme greater than equine lysozyme (KD approximately 5 x 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (KD = 3 x 10(-3) M) was obtained for bovine alpha-lactalbumin, but not for the other proteins studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural comparison between oxidized and reduced Escherichia coli thioredoxin. Proton NMR and CD studies.

Escherichia coli thioredoxin (Mr 11,700) usually functions as a hydrogen carrier protein that undergoes reversible oxidation/reduction reactions of its active-site disulfide linkage. By use of a number of assigned and identified resonances in one- and two-dimensional 1H NMR spectra, the two forms of the protein have been compared. Only groups that are relatively close to the active-site Cys-32, Cys-35 linkage such as Trp-28, Trp-31, Phe-27, Ala-29, and Val-25 undergo substantial changes in their 1H NMR chemical shift upon reduction. Various residues that are further removed from the active site, like Tyr-49, Tyr-70, His-6, Phe-12, Phe-81, and Phe-102, appear to be little affected (less than 0.02 ppm) by the reduction, suggesting that the rest of the protein structure is not much affected. Thus, the structural changes that occur upon reduction appear to be localized to the disulfide-containing turn and the central strand of the twisted beta-sheet that directly leads to this turn. Notwithstanding the apparent similarity in the secondary and tertiary structures of the oxidized and reduced forms of the protein, the thermal stability of the protein decreases by 10 degrees C upon the reduction of the single disulfide. This was found by both 1H NMR and near- and far-ultraviolet circular dichroism studies. Oxidized thioredoxin was also more resistant to alkaline denaturation. Furthermore, the exchange rate of the relatively stable slow-exchanging backbone amide protons that are part of the core of the twisted five-stranded beta-sheet of thioredoxin increases substantially after reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear magnetic resonance studies on calmodulin: calcium-induced conformational change.

The 400-MHz 1H nuclear magnetic resonance (NMR) studies were carried out on the Ca2+-induced conformational change of calmodulins (CaM's) isolated from scallop testis and pig brain. The resonances were found to be classified approximately into three groups. The resonances of group I, which are perturbed by the binding of Ca2+ to the high-affinity sites, include those of tyrosine-138, epsilon-trimethyllysine-115, histidine-107, tyrosine-99, etc. The previous assignments for tyrosine- (Tyr) 138 [Seamon, K. B. (1980) Biochemistry 19, 207] were corrected. The resonances of group II, which are affected by the binding of Ca2+ to the low-affinity sites, include those of a phenylalanine (Phe), a high field shifted methyl, and a low field shifted alpha-methine. Group III (related to the binding of Ca2+ to both the high-and low-affinity sites) includes the resonances of a Phe, a high field shifted methyl, and threonine-143. It is concluded that sites III and IV are the high-affinity sites. The off-rate of Ca2+ from the high-affinity sites is slower than 50 s-1 while the off-rate from the low-affinity sites is faster than 600 s-1. In the Ca2+-free state, there exists a hydrophobic region containing three phenylalanine (probably Phe-89, Phe-92, and Phe-141), a valine, and an isoleucine in the vicinity of sites III and IV. Tyr-138 is distant from these amino acids. Upon binding of Ca2+ to the high-affinity sites, one of the Phe residues and the valine approach Tyr-138. Similar structural changes were observed between CaM and troponin C when Ca2+ ions are bound to the high-affinity sites. CaM changes in a somewhat different way from troponin C when Ca2+ ions are bound to the low-affinity sites.

Nuclear magnetic resonance studies on calmodulin: spectral assignments in the calcium-free state.

The 400-MHz proton magnetic resonance spectra of calcium-free scallop testis calmodulin (CaM) and pig brain CaM were observed. Detailed spectral assignments were made by resolution enhancement, spin decoupling, and nuclear Overhauser enhancement (NOE) experiments as well as pH titration. Comparison between spectra of scallop testis CaM and pig brain CaM were also utilized for the assignment. Previous assignments for tyrosine-99, histidine-107, epsilon-trimethyllysine-115, and tyrosine-138 [Seamon, K. B. (1980) Biochemistry 19, 207; Krebs, J., & Carafoli, E. (1982) Eur. J. Biochem. 124, 619] were confirmed. Phenylalanine-99 and threonine-143 of scallop testis CaM were identified. Sixteen methyl resonances from one isoleucine, two valines, nine methionines, and the amino-terminal acetyl group were identified. First-stage assignments were made of resonances arising from seven phenylalanines. The uniquely high field shifted phenylalanine resonance previously reported by Seamon was found to consist of two doublets from the two pairs of delta protons of two phenylalanines. The NOE experiments showed that the two phenylalanines are located closely to each other. The large high-field shifts of these phenylalanines were accounted for the ring-current effects due to their proximity. An isoleucine and a valine of which methyl resonances appear at high fields were found to be situated closely to each other. It was found that two delta protons and two epsilon protons of almost all aromatic residues are magnetically equivalent, suggesting that the local structure of aromatic residues is so flexible as to permit the rapid flipping motion of the ring.

Fourier-transform infrared spectroscopic studies on the coordination of the side-chain COO- groups to Ca2+ in equine lysozyme.

Interactions between Ca2+ and the Asp side chains in the Ca2+-binding site of equine lysozyme were investigated by Fourier-transform infrared (FT-IR) spectroscopy. In the spectrum of equine lysozyme, the intensities of the bands at about 1595 cm-1 and 1578 cm-1 in the region of the COO antisymmetric stretches increased upon Ca2+ binding. In the region of the COO- symmetric stretches, the loss of intensity at about 1388 cm-1 and gains of intensities at about 1423 cm-1 and 1403 cm-1 were observed due to Ca2+ binding to equine lysozyme. The spectral changes for equine lysozyme indicate that the COO- groups of Asp85, Asp90 and Asp91 in the Ca2+-binding site coordinate to Ca2+ in the pseudo-bridging mode, where divalent metal cation is bound to one of the two oxygens in the COO- group and a water molecule is hydrogen bonded to the other oxygen. The results presented here provide further evidence for a high degree of similarity between Ca2+-binding lysozyme and alpha-lactalbumin. The effects of Ca2+ binding on the main-chain conformation of equine lysozyme were compared with those of bovine alpha-lactalbumin and hen egg-white lysozyme.