Hydrogen exchange of the tryptophan residues in bovine, goat, guinea pig, and human alpha-lactalbumin

Hydrogen exchange of the individual tryptophan residues of bovine, goat, guinea pig, and human alpha-lactalbumin has been studied by both ultraviolet and NMR spectra. The assignment of the slowly exchanging imino proton resonances to the tryptophan residues (Trp26 and Trp60) was obtained by comparison of the nuclear Overhauser effect difference spectra of bovine, guinea pig, and human alpha-lactalbumin. Taking account of the thermal unfolding of each alpha-lactalbumin, the hydrogen exchange rates of the individual tryptophan residues are analyzed. The temperature dependence of the exchange rates classified their exchange mechanisms into two exchange processes: the "low activation energy process" and the "high activation energy process" which is associated directly with the global thermal unfolding of the protein. Trp26 of alpha-lactalbumin exchanges through the high activation energy process. The exchange behavior of Trp26 of guinea pig alpha-lactalbumin suggests a difference of the globally unfolded state of the protein from the other species. The exchange mechanism of Trp60 of human alpha-lactalbumin is the low activation energy process in contrast with those of the bovine and goat proteins, although their global thermodynamic properties are similar to each other. Trp104 and Trp118 of alpha-lactalbumin exchange through the low activation energy process, and the reaction rates are affected by the local structural differences around the tryptophan residues among these proteins. The results presented in this paper indicate that the hydrogen exchange rate through the low activation energy process provides the information only about the local nature of a protein while that through the high activation energy process provides the information about the global nature of a protein.     

Hydrogen-deuterium exchange in the histidine residues of bovine alpha-lactalbumin.

The proton magnetic resonance spectrum of bovine alpha-lactalbumin has been observed, and three peaks assignable to the position-2 CH protons of the three histidine rpsidues (His 32, 68, and 107) of this protein have been subjected to detailed examination. The assignments of these peaks to His 32, 68, and 107 were made on the basis of the difference in their reactivities with iodoacetic acid. The rate constants of the hydrogen-deuterium exchange reactions were found to be 8.0 X 10(-5), 2.6 X 10(-4), and 8.0 X 10(-5) min-1, respectively, at pH 8.5 and at 35 degrees, while at 62 degrees all three were found to be 0.84 approximately 1.1 X 10(-2) min-1. On the basis of these data, it has been shown that, in the native form of this protein, His 68 is the most exposed to the solvent while His 32 and His 107 are buried slightly deeper in the surface of the molecule. The fluctuation amplitudes gamma, or the effective chances of His 32, 68, and 107 to be fully exposed to the solvent, were found to be 0.4, 1.3, and 0.4, respectively.     

Hydrogen exchange at the core of Escherichia coli alkaline phosphatase studied by room-temperature tryptophan phosphorescence

The room-temperature tryptophan (Trp) phosphorescence lifetime is sensitive to details of the local environment and has been shown to increase significantly in some proteins following H-D exchange. Careful analysis of the phosphorescence lifetime distribution of Trp 109 in Escherichia coli alkaline phosphatase (AP) in solution as a function of time during the H-D exchange shows that this process corresponds to a two-state reaction resulting from the deuteration of a single, specific hydrogen in the core of the protein. The absence of a pH dependence of the exchange rate suggests that the exchange is not an EX2 process, and therefore, a certain degree of unfolding is required for exchange to occur. This discovery opens up the use of phosphorescence-detected hydrogen exchange as a sensitive tool for monitoring the local susceptibility and activation energy for exchange in proteins having a phosphorescent Trp and, for example, for studying the effects of local mutations upon that susceptibility.     

Hydrogen-deuterium exchange of the tryptophan residues in bovine α-lactalbumin

Effects of deuteration on the Raman spectrum of a tryptophan residue have been examined. The 1386 cm^?1 line of deuterated tryptophan residue has been found to be useful for tracing the hydrogen-deuterium exchange reaction of this residue in a protein. An examination on bovine α-lactalbumin at pH 6.4 and at 20°C indicates that two of the four tryptophan residues exchange with a rate constant much greater than 9 × 10^?4 sec^?1, while the other two exchange with a rate constant of 4 × 10^?5 sec^?1. The latter two have been assigned to Trp 28 and Trp 108 of this protein. The kinetics of hydrogen-deuterium exchange reaction of completely “free” tryptophan residue have been examined by a proton magnetic resonance study on tryptophan itself. By taking the result of this examination into account, the chance of exposure to the solvent for Trp 28 or Trp 108 has been estimated to be 3 × 10^?6 at pH 6.4 and at 20°C.     

The membrane-bound conformation of alpha-lactalbumin studied by NMR-monitored 1H exchange

The interaction of bovine alpha-lactalbumin (BLA) with negatively charged phospholipid bilayers was studied by NMR monitored 1H exchange to characterize the conformational transition that enables a water-soluble protein to associate with and partially insert into a membrane. BLA was allowed to exchange in deuterated buffer in the absence (reference) and the presence (membrane-bound) of acidic liposomes at pH 4.5, experimental conditions that allow efficient protein-membrane interaction. After adjusting the pH to 6.0, to dissociate the protein from the membrane, reference and membrane-released samples of BLA were analysed by (F1) band-selective homonuclear decoupled total correlation spectroscopy in the alphaH-NH region. The overall exchange behaviour of the membrane-bound state is molten globule-like, suggesting an overall destabilization of the polypeptide. Nevertheless, the backbone amide protons of residues R10, L12, C77, K94, K98, V99 and W104 show significant protection against solvent exchange in the membrane-bound protein. We propose a mechanism for the association of BLA with negatively charged membranes that includes initial protonation of acidic side-chains at the membrane interface, and formation of an interacting site with the membrane which involves helixes A and C. In the next step these helices would slide away from each other, adopting a parallel orientation to the membrane, and would rotate to maximize the interaction between their hydrophobic residues and the lipid bilayer.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

Proton-magnetic-resonance spectroscopic study of the histidine residues of bovine alpha-lactalbumin.

A study of the three histidine residues of bovine alpha-lactalbumin has been made using proton magnetic resonance (PMR) spectroscopy in order to obtain information on their environments in the protein and thereby to test in part the previously proposed structure. PMR titration curves are obtained for the H-4 resonances using difference spectroscopy and for the H-2 resonances and the 1-H-2-H exchange rates of the H-2 protons have been measured. The assignment of resonances to particular histidine residues is achieved by utilising their selective reaction with iodoacetate in conjunction with a PMR study of the carboxymethylation of alpha-N-acetyl-L-histidine. The H-2 and H-4 resonances labelled 1, 2 and 3 starting from the downfield end of the spectrum are assigned to histidine residues 107, 68 and 32 respectively. Their apparent pK values at low ionic strength and 20 degrees C are 5.78, 6.49 and 6.51 respectively. The experimental results on two histidine residues are consistent with the predictions of the proposed structure, which indicate that histidine-68 is an external residue and histidine-32 is partially buried and in the vicinity of aromatic residues. The experimental data on histidine 107 can also be rationalised with less certainty in terms of the proposed structure, which indicates a partially buried residue that may be involved in hydrogen bonding.     

Membrane binding of MARCKS-related protein studied by tryptophan fluorescence spectroscopy

MARCKS-related protein (MRP) is a peripheral membrane protein whose binding to membranes is mediated by the N-terminal myristoyl moiety and a central, highly basic effector domain. MRP mediates cross-talk between protein kinase C and calmodulin and is thought to link the actin cytoskeleton to the plasma membrane. Since MRP contains no tryptophan residues, we mutated a phenylalanine in the effector domain to tryptophan (MRP F93W) and used fluorescence spectroscopy to monitor binding of the protein to phospholipid vesicles. We report in detail the evaluation procedure necessary to extract quantitative information from the raw data. The spectra of MRP F93W obtained in the presence of increasing amounts of lipid crossed at an isosbestic point, indicating a simple transition between two states: free and membrane-bound protein. The change in fluorescence toward values typical of a more hydrophobic environment was used to quantify membrane binding. The partition coefficient agreed well with values obtained previously by other methods. To study the interaction of the N-terminus of MRP with membranes, a tryptophan residue was also introduced at position 4 (MRP S4W). Our data suggest that only the myristoylated N-terminus interacted with liposomes. These results demonstrate the versatility of site-directed incorporation of tryptophan residues to study protein-membrane interactions.     

Torsional vibrational modes of tryptophan studied by terahertz time-domain spectroscopy

The low-frequency torsional modes, index of refraction, and absorption of a tryptophan film and pressed powders from 0.2 to 2.0 THz (6.6-66 cm(-1)) were measured by terahertz time-domain spectroscopy at room temperature. It was found that there were two dominated torsional vibrational modes at around 1.435 and 1.842 THz. The associated relaxation lifetimes ( approximately 1 ps) for these modes of the tryptophan molecule were measured. Using a density-functional calculation, the origins of the observed torsional vibrations were assigned to the chain and ring of the tryptophan molecule.     

Perturbation of tryptophan residues by point mutations in bacteriophage T4 lysozyme studied by optical detection of triplet-state magnetic resonance spectroscopy

We have investigated perturbations of the triplet-state properties of Trp residues in bacteriophage T4 lysozyme caused by point mutations using low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy. Five temperature-sensitive mutants have been studied in detail. These include lysozymes with the point mutations Gln-105----Ala, Gln-105----Gly, Gln-105----Glu, Ala-146----Thr, and Trp-126----Gln. Changes in phosphorescence 0,0 band wavelength, intensity, the triplet-state zero-field splitting (ZFS), and the wavelength dependence of the ZFS were detected only from Trp-138 in each mutant. In the case of the Q105A mutation, the perturbations on Trp-138 have been ascribed to the combination of an increase in the polarizability of the environment and to the loss of hydrogen bonding of the enamine nitrogen of indole. For the Q105G mutation, we believe that Q is replaced by a solvent molecule in H bonding, leading to relatively small changes. In the Q105E mutation, the perturbation results largely from the introduction of a charged residue. In the case of the mutation A146T, the perturbation is associated with a local conformational change in which Trp-138 is shifted to a more solvent-exposed location. On the other hand, no significant spectroscopic changes in Trp-126 and Trp-158 were found in any of the mutants, suggesting that the perturbations are probably localized near Trp-138 for the mutations of positions 105 and 146. However, in the mutation W126Q, which occurs approximately 16 A away from Trp-138, significant changes of Trp-138 are detected, suggesting that the effects of this mutation are propagated over large distances.     

Application of the pH-jump method to the titration of tyrosine residues in bovine alpha-lactalbumin.

A stopped-flow technique has been developed for the zero-time spectrophotometric titration of tyrosine residues in the purely native or in the purely alkaline denatured state of alpha-lactalbumin that undergoes an alkaline conformational transition in the pH region of tyrosine ionization. The progressive absorption change at 298 nm caused by a pH jump from neutral pH is shown to result from the change in ionization of the tyrosine residues brought about by a first-order process of the conformational transition. Extrapolation to zero time gives the titration curve for purely native alpha-lactalbumin. Similarly, the pH jump from highly alkaline pH gives the titration curve for the purely alkaline denatured protein. The method should be generally applicable to other proteins that contain tyrosines. Analysis of the titration curves suggests that the four tyrosines in native alpha-lactalbumin have pK values of 10.5, 11.8, 11.8, and 12.7, respectively. After the alkaline transconformation, all of them become titrated normally with a pK value of 10.3. A comparison of these results with the ionization behavior of tyrosines in hen egg white and human lysozymes is presented and discussed in terms of differences in the sequences of the proteins.     

Comparison of the structural and dynamical properties of holo and apo bovine alpha-lactalbumin by NMR spectroscopy

In the presence of 0.5 M NaCl at pH 7.1, the Ca(2+)-free apo form of recombinant bovine alpha-lactalbumin (BLA) is sufficiently stabilised in its native state to give well-resolved NMR spectra at 20 degrees C. The (1)H and (15)N NMR resonances of native apo-BLA have been assigned, and the chemical-shifts compared with those of the native holo protein. Large changes observed between the two forms of BLA are mainly limited to the Ca(2+)-binding region of the protein. These data suggest that Na(+) stabilises the native apo state through the screening of repulsive negative charges, at the Ca(2+)-binding site or elsewhere, rather than by a specific interaction at the vacant Ca(2+)-binding site. The hydrogen exchange protection of residues in the Ca(2+)-binding loop and the C-helix is reduced in the apo form compared to that in the holo form. This indicates that the dynamic behaviour of this region of the protein is substantially increased in the absence of the bound Ca(2+). Real-time NMR experiments show that the rearrangements of the structure associated with the conversion of the holo to apo form of the protein do not involve the detectable population of partially unfolded intermediates. Rather, the conversion appears to involve local reorganisations of the structure in the vicinity of the Ca(2+)-binding site that are coupled to the intrinsic fluctuations in the protein structure.     

On the reactivities of the tryptophan residues of human alpha-lactalbumin to 2-hydroxy-5-nitrobenzyl bromide.

The reaction of human alpha-lactalbumin with the tryptophan reagent 2-hydroxy-5-nitrobenzyl bromide has been studied. This protein has 3 tryptophan residues (Trp-60, Trp-104 and Trp-118) all of which are accessible to the reagent at pH 2.7 or 7. Trp-60 of human alpha-lactalbumin is much more reactive than Trp-60 of bovine alpha-lactalbumin (Barman, T. E. (1972) Biochim. Biophys. Acta 257, 297-313). As with bovine alpha-lactalbumin, at pH 2.7, 2-hydroxy-5-nitrobenzyl bromide is specific for tryptophan but at pH 7 His-32 also reacts. When treated with the tryptophan reagent, both alpha-lactalbumins lose their specifier protein activities in the lactose synthase (UDPgalactose:D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) reaction.     

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)     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.