Fourier-transform infrared spectroscopy study of the pressure-induced changes in the structure of the bovine alpha-lactalbumin: the stabilizing role of the calcium ion.

The Fourier-transform infrared spectroscopy (FTIR) technique with a diamond anvil cell has been applied for examination of the pressure-induced changes occurring in the secondary structure of the alpha-lactalbumin. This is the first high-pressure FTIR study of a calcium-binding protein which simultaneously takes into account spectral changes in both the calcium-ion-binding carboxyl groups' band and the amide I/I' vibrational band. Spectral behavior of three kinds of the protein: the undeuterated holoform, the fully deuterated holoform, and the undeuterated apoform was compared in the pressure range from 0.1 MPa up to 740 MPa. We found that the binding of calcium remarkably stabilizes the alpha-lactalbumin against pressure as it is followed approximately by a 200-MPa increase of the value of pressure at which denaturation occurs. A quantitative analysis of the band of antisymmetrical stretching vibrations of the calcium-binding carboxyl groups revealed that the pressure-induced changes in the calcium-binding loop occur in two stages. Binding of the calcium ion seemingly increases the pressure-stability of the calcium-binding loop to a higher degree than the pressure-stability of the secondary structure of the alpha-lactalbumin. We have also discussed in detail the complex pressure-enhanced H/D exchange in the alpha-lactalbumin. Finally, we have proposed a new assignment of major peaks in the helical region of the amide I/I' spectral band of the partially deuterated alpha-lactalbumin.     

Binding strength of calcium ion to bovine alpha-lactalbumin

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.     

A calorimetric study of the influence of calcium on the stability of bovine alpha-lactalbumin

Bovine alpha-lactalbumin has been studied by differential scanning calorimetry with various concentrations of calcium to elucidate the effect of this ligand on its thermal properties. In the presence of excess calcium, alpha-lactalbumin unfolds upon heating with a single heat-absorption peak and a significant increase of heat capacity. Analysis of the observed heat effect shows that this temperature-induced process closely approximates a two-state transition. The transition temperature increases in proportion with the logarithm of the calcium concentration, which results in an increase in the transition enthalpy as expected from the observed heat capacity increment of denaturation. As the total concentration of free calcium in solution is decreased below that of the proteins, there are two temperature-induced heat absorption peaks whose relative area depends on the calcium concentration, such that further decrease of calcium concentration results in a increase of the low-temperature peak and a decrease of the high-temperature one. The high-temperature peak occurs at the same temperature as the unfolding of the holo-protein, while the low-temperature peak is within the temperature range associated with the unfolding of the apo-protein. Statistical thermodynamic modeling of this process shows that the bimodal character of the thermal denaturation of bovine alpha-lactalbumin at non-saturated calcium concentrations is due to a high affinity of Ca2+ for alpha-lactalbumin and a low rate of calcium exchange between the holo- and apo-forms of this protein. Using calorimetric data, the calcium-binding constant for alpha-lactalbumin has been determined to be 2.9 x 10(8) M-1.     

Anti-aggregation properties of trehalose on heat-induced secondary structure and conformation changes of bovine serum albumin

During our experimental work, aggregation of bovine serum albumin was obtained incubating the protein solution at 60 °C to investigate temperature-induced secondary structure, conformation changes and anti-aggregative activity of trehalose. IR-measurements suggested that in the presence of 1.0 M of trehalose there is a little increase in short segment connecting ?α-helical and a clearly decrease in the loss of ?α-helix structure and in the formation of intermolecular and antiparellel β-sheet up to 78 and 55%, respectively. Useful information also arose following the temperature evolution of Amide I′ band profile in the range of temperature between 25 and 90 °C in absence or in presence of 1.0 M trehalose. Complementary information is obtained by electrophoresis, circular dichroism, fluorescence spectroscopy, titration of SH groups and light scattering measurements. Results encouraged biotechnology and pharmaceutical application of the disaccharide and provided evidence for its utilization in degenerative diseases evolving via aggregation process.     

Effect of metal ion binding on the secondary structure of bovine alpha-lactalbumin as examined by infrared spectroscopy

We have examined the influence of monovalent and divalent cations on the secondary structure of bovine alpha-lactalbumin at neutral pH using Fourier-transform infrared spectroscopy. Our present studies are based on previously reported amide I' component band assignments for this protein [Prestrelski, S. J., Byler, D. M., & Thompson, M. P. (1991) Int. J. Pept. Protein Res. 37, 508-512]. The results indicate that upon dissolution, alpha-lactalbumin undergoes a small, but significant, time-dependent conformational change, regardless of the ions present. Additionally, these studies provide the first quantitative measure of the well-known secondary structural change which accompanies calcium binding. Results indicate that removal of Ca2+ from holo alpha-lactalbumin results in local unfolding of the Ca(2+)-binding loop; the spectra indicate that approximately 16% of the backbone chain changes from a rigid coordination complex to an unordered loop. We have also examined the effects of binding of several other metal ions. Our studies have revealed that binding of Mn2+ to apo alpha-lactalbumin (Ca(2+)-free), while inducing a small, but significant, conformational change, does not cause the alpha-lactalbumin backbone conformation to change to that of the holo (Ca(2+)-bound) form as characterized by infrared spectroscopy. Similar changes to those induced by Mn2+ are observed upon binding of Na+ to apo alpha-lactalbumin, and furthermore, even at very high concentrations (0.2 M), Na+ does not stabilize a structure similar to the holo form. Binding of Zn2+ to the apo form of alpha-lactalbumin does not result in significant backbone conformational changes, suggesting a rigid Zn(2+)-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study on bovine alpha-lactalbumin and lysozyme by nanosecond fluorometry.

Analysis of the time decay of fluorescence anisotropy of 1-dimethylaminoaphthalene-5-sulfonyl (DNS) and fluorescamine derivatives of bovine alpha-lactalbumin and lysozyme reveals that no significant differences in mean rotational relaxation times are present. While fluorescamine molecules appear to orient randomly on these proteins, DNS is bound with a preferential orientation. Other fluorescence characteristics of the labels are also cited.     

A protein dissection study demonstrates that two specific hydrophobic clusters play a key role in stabilizing the core structure of the molten globule state of human alpha-lactalbumin

The molten globule state of alpha-lactalbumin (alpha LA) has served as a paradigm for understanding the role of these partially folded states in protein folding. We previously showed that a peptide construct consisting of the A and B helices (residues 1-38) cross-linked to the D- and C-terminal 3(10) helices (residues 101-120) of alpha LA is capable of folding to a stable molten globule-like state. Here, we report the study of three peptide constructs that are designed to investigate the contribution two short hydrophobic sequences located near the C-terminus of alpha LA make to the structure and stability of the alpha LA molten globule state. These regions of the protein have been shown to form stable non-native structures in isolation. The three peptide constructs contain residues 1-38 cross-linked to three separate C-terminal peptides via the native 28-111 disulfide bond. The C-terminal peptides consist of residues 101-114, 106-120, and 106-114. The results of CD, fluorescence, ANS binding, and urea denaturation experiments indicate that constructs that lack either of the hydrophobic sequences (residues 101-105 and 115-120) are significantly less structured. These results highlight the importance of long-range, mutually stabilizing interactions within the molten globule state of the protein. Proteins 2001;42:237-242.     

Dissection of heat-induced systemic signals: superiority of ion fluxes to voltage changes in substomatal cavities

Using non-invasive ion-selective microprobes, that were placed in substomatal cavities, long-distance signalling has been investigated in intact Hordeum vulgare and Vicia faba seedlings. Heat (flame), applied to one leaf (S-leaf), triggers apoplastic ion activity (pH, pCa, pCl) transients in a distant leaf (T-leaf), all largely independent of simultaneously occurring action potential-like voltage changes. While apoplastic pCa and pH increase (Ca²⁺-, H⁺-activities decrease), pCl decreases (Cl⁻-activity increases). As the signal transfer from the S- to the T-leaf is too fast to account for mass flow, the heat-induced pressure change is primarily responsible for changes in voltage (H⁺ pump deactivation) as well as for the ion fluxes. The pCa transient precedes the pCl- and pH responses, but not the voltage change. Since the apoplastic pCl decrease (Cl⁻ increase) occurs after the pCa increase (Ca²⁺ decrease) and after the depolarization, we argue that the Cl⁻ efflux is a consequence of the Ca²⁺ response, but has no part in the depolarization. Kinetic analysis reveals that pH- and pCl changes are interrelated, indicated by the action of the anion channel antagonist NPPB, which inhibits both pCl- and pH changes. It is suggested that efflux of organic anions into the apoplast causes the pH increase rather than the deactivation of the plasma membrane H⁺ pump. Since there is considerably more information in ion activity changes than in a single action- or variation potential and heat-induced ion fluxes occur more reliably than voltage changes, released by milder stimuli, they are considered systemic signalling components superior to voltage.     

FTIR study of secondary structure changes in Epidermal Growth Factor by gold nanoparticle conjugation

Conformation of protein is vital to its function, but may get affected when processing to manufacture products. It is therefore important to understand structural changes during each step of production. In this study, we investigate secondary structure changes in the targeting protein Epidermal Growth Factor (EGF) during synthesis of theranostic bifunctional nanoparticle, devised for Photodynamic therapy of breast cancer. We acquired FTIR spectra of EGF; unconjugated, post treatment with α-lipoic acid, attached to gold nanoparticle, and bound to the bifunctional nanoprobe. We observed decreasing disordered structures and turns, and increasing loops, as the synthesis process progressed. There was an overall increase in β-sheets in final product compared to pure EGF, but this increase was not linear and fluctuated. Previous crystal structure studies on EGF-EGFR complex have shown loops and β-sheets to be important in the binding interaction. Since our study found increase in these structures in the final product, no adverse effect on binding function of EGF was expected. This was confirmed by functional assays. Such studies may help modify synthesis procedures, and thus secondary structures of proteins, enabling increased functionality and optimum results.     

Effects of 50 Hz magnetic fields on fMLP-induced shape changes in invertebrate immunocytes: The role of calcium ion channels

Plasma membrane Ca(2+) channels in immunocytes from the mussel Mytilus galloprovincialis exposed to 50 Hz sine wave magnetic fields (MFs) of various strengths were studied. At levels of 300 microT and above, MFs reduce shape changes in immunocytes induced by the chemotactic substance N-formyl-Meth-Leu-Phe, and this effect involves L-type Ca(2+) channels. Upon the addition of the Ca(2+) blocker verapamil to molluscan immunocytes exposed to MFs results in a synergistic cytotoxic action, while in the presence of the Ca(2+) opener SDZ-202, 791, a reactivation of the cells is observed. This suggests that, as previously reported for potassium channels, the damage to Ca(2+) channels induced by short exposure to MF at appropriate intensities is not permanent.     

Effects of the stabilization of the molten globule state on the folding mechanism of alpha-lactalbumin: a study of a chimera of bovine and human alpha-lactalbumin

The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.     

Effects of bovine alpha-lactalbumin on gastric defense mechanisms in naive rats

We recently investigated the effects of the major proteins in cow's milk on gastric mucosal injuries in rat ulcer models. We found that alpha-lactalbumin (alpha-LA) has marked preventive effects against gastric mucosal injuries and that prostaglandin (PG) synthesis may contribute to these effects [Matsumoto et al., Biosci. Biotechnol. Biochem., 65, 1104-1111, 2001]. In this study, we investigated the effects of alpha-LA on several defense mechanisms of gastric mucosa by evaluating gastric PGE2 content, gastric mucin content, gastric luminal pH, gastric fluid volume, and gastric emptying in naive rats. Oral administration of alpha-LA (200, 500, and 1000 mg/kg) elevated endogenous PGE2 levels in gastric tissue and increased the gastric mucin contents of both the gastric fluid and the adherent mucus gel layer. In addition to these PG-related responses, alpha-LA also caused PG-independent responses such as elevation of gastric luminal pH, increase in gastric fluid volume, and delay in gastric emptying. These responses were observed to be dose-dependent (200-1000 mg/kg of alpha-LA). Thus, we demonstrated that alpha-LA enhances both PG-dependent and PG-independent gastric defense mechanisms in naive rats. Both of these mechanisms are probably involved in its gastroprotective action.     

The role of free calcium ion in calcium release in skinned muscle fibers

Major questions in excitation--contraction coupling of fast skeletal muscle concern the mechanism of signal transmission between sarcolemma and sarcoplasmic reticulum (SR), the mechanism of SR Ca release, and operation of the SR active transport system during excitation. Intracellular Ca movement can be studied in skinned muscle fibers with more direct control, analysis of 45Ca flux, and simultaneous isometric force measurements. Ca release can be stimulated by bath Ca2+ itself, ionic "depolarization," Mg2+ reduction, or caffeine. The effectiveness of bath Ca2+ has suggested a possible role for Ca2+ in physiological release, but this response is difficult to analyze and evaluate. Related evidence emerged from analysis of other responses: with all agents studied, stimulation of 45Ca efflux is highly Ca2+-dependent. The presence of a Ca chelator prevents detectable stimulation by ionic "depolarization" or Mg2+ reduction and inhibits the potent caffeine stimulus; inhibition is graded with chelator concentration and caffeine concentration, and is synergistic with inhibition by increased Mg2+. The results indicate that a Ca2+-dependent pathway mediates most or all of stimulated 45Ca efflux in skinned fibers, and has properties compatible with a function in physiological Ca release.     

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study.

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.