Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.     

X-ray structural evidence for a local helix-loop transition in alpha-lactalbumin.

The three-dimensional structure of human alpha-lactalbumin for two crystal forms has been determined by x-ray analysis. One crystal (the form LT) was obtained at pH 4.2 and room temperature, while the other crystal (the form HT) was grown at pH 6.5 and 37 degrees C. The backbone structure for Lys1-Ile95 residues is almost conserved between the two structures as indicated by the root mean square difference of 0.30 A for the superposition of equivalent C alpha atoms. The calcium ion is surrounded by seven oxygen atoms of three carboxyl groups, two carbonyl groups, and two water molecules, which form a distorted pentagonal bipyramid in both structures. A large difference in polypeptide folding is found in the region of Leu96-Leu123 residues. Especially in the region of Trp104-Cys111 residues, a distorted alpha-helix is observed in the form HT while a loop structure is formed in the other crystal. The fact that the crystals of both forms appeared in the same batch at pH 6.5 and room temperature indicates that the human alpha-lactalbumin structure is highly fluctuated in solution and the folding and unfolding of the alpha-helix of Trp104-Cys111 residues are in equilibrium. Since the crystal of the form HT exclusively appeared around the physiological temperature, the structure of this form can be considered as the native structure. The partially unfolded structure in the form LT indicates that the local denaturation occurs even at room temperature.     

Isolation and identification of three bactericidal domains in the bovine alpha-lactalbumin molecule

Proteolytic digestion of alpha-lactalbumin by pepsin, trypsin and chymotrypsin yielded three polypeptide fragments with bactericidal properties. Two fragments were obtained from the tryptic digestion. One was a pentapeptide with the sequence EQLTK (residues 1-5) and the other, GYGGVSLPEWVCTTF ALCSEK (residues (17-31)S-S(109-114)), was composed of two polypeptide chains held together by a disulfide bridge. Fragmentation of alpha-lactalbumin by chymotrypsin yielded CKDDQNPH ISCDKF (residues (61-68)S-S(75-80)), also a polypeptide composed of two polypeptide chains held together by a disulfide bridge. The three polypeptides were synthesized and found to exert antimicrobial activities. The polypeptides were mostly active against Gram-positive bacteria. Gram-negative bacteria were only poorly susceptible to the bactericidal action of the polypeptides. GYGGVSLPEWVCTTF ALCSEK was most, EQLTK least bactericidal. Replacement of leucine (23) with isoleucine, having a similar chemical structure but higher hydrophobicity, in the sequence GYGGVSLPEWVCTTF ALCSEK significantly reduced the bactericidal capacity of the polypeptide. Digestion of alpha-lactalbumin by pepsin yielded several polypeptide fragments without antibacterial activity. alpha-Lactalbumin in contrast to its polypeptide fragments was not bactericidal against all the bacterial strains tested. Our results suggest a possible antimicrobial function of alpha-lactalbumin after its partial digestion by endopeptidases.     

Crystallography of alpha-lactalbumin. III. Crystals of baboon milk alpha-lactalbumin.

Crystals of baboon α-lactalbumin suitable for structure analysis at high resolution have been obtained. They have cell dimensions $\text{a = 35.5}\text{A}\text{?}\text{, b = 69.1}\text{A}\text{?}\text{, c = 46.1}\text{A}\text{?}$, space group P2₁2₁2 and contain one molecule of α-lactalbumin per asymmetric unit.     

Gamma radiation effects on alpha-lactalbumin: structural modifications

Alpha-lactalbumin was irradiated in the lyophilized state in air at ambient temperature. The irradiated protein was examined by size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, circular dichroism, and microcalorimetry. Irradiation induced the loss of aromatic amino acids and of helicity so that fragmentation and aggregation products were obtained. The thermodynamic properties of the protein were also modified. The irradiated protein had lower stability, however, the temperature at which denaturation occurred process remained constant.     

Rapid collapse and slow structural reorganisation during the refolding of bovine alpha-lactalbumin.

The refolding of bovine alpha-lactalbumin (BLA) from its chemically denatured state in 6 M GuHCl has been investigated by a variety of complementary biophysical approaches. CD experiments indicate that the species formed in the early stages of refolding of the apo-protein have at least 85 % of the alpha-helical content of the native state, and kinetic NMR experiments show that they possess near-native compactness. Hydrogen exchange measurements using mass spectrometry and NMR indicate that persistent structure in these transient species is located predominantly in the alpha-domain of the native protein and is similar to that present in the partially folded A-state formed by the protein at low pH. The extent of the exchange protection is, however, small, and there is no evidence for the existence of well-defined discrete kinetic intermediates of the type populated in the refolding of the structurally homologous c-type lysozymes. Rather, both mass spectrometric and NMR data indicate that the rate-determining transition from the compact partially structured (molten globule) species to the native state is highly cooperative. The data show that folding in the presence of Ca2+ is similar to that in its absence, although the rate is increased by more than two orders of magnitude. Sequential mixing experiments monitored by fluorescence spectroscopy indicate that this slower folding is not the result of the accumulation of kinetically trapped species. Rather, the data are consistent with a model in which binding of Ca2+ stabilizes native-like contacts in the partially folded species and reduces the barriers for the conversion of the protein to its native state. Taken together the results indicate that folding of BLA, in the presence of its four disulphide bonds, corresponds to one of the limiting cases of protein folding in which rapid collapse to a globule with a native-like fold is followed by a search for native-like side-chain contacts that enable efficient conversion to the close packed native structure.     

A procedure for detecting structural domains in proteins.

A procedure is described for detecting domains in proteins of known structure. The method is based on the intuitively simple idea that each domain should contain an identifiable hydrophobic core. By applying the algorithm described in the companion paper (Swindells MB, 1995, Protein Sci 4:93-102) to identify distinct cores in multi-domain proteins, one can use this information to determine both the number and the location of the constituent domains. Tests have shown the procedure to be effective on a number of examples, even when the domains are discontinuous along the sequence. However, deficiencies also occur when hydrophobic cores from different domains continue through the interface region and join one another.     

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.     

The structural basis of ankyrin function. I. Identification of two structural domains

The structure of ankyrin, a major linking protein between spectrin and the erythrocyte membrane, was analyzed after restricted proteolytic digestion at 0 degree C. By the use of two-dimensional peptide mapping, we found that tryptic digestion of ankyrin (1 h, 0 degree C) resulted in the production of two nonoverlapping peptides of molecular weights 82,000 and 55,000. The 82,000-dalton peptide had a basic isoelectric point (7.9) and was remarkably sensitive to further proteolytic digestion; after 24 h at 0 degree C, trypsin completely digested this peptide into fragments too small to detect by gel electrophoresis. The 55,000-dalton peptide was neutral (isoelectric point = 6.9-7.2) and more resistant to further proteolytic cleavage. After a 24-h digestion with trypsin at 0 degrees C, the 55,000-dalton peptide was cleaved into two complementary fragments of molecular weight 32,000 and 15,000. Analysis of phosphorylated ankyrin indicated that the phosphates were exclusively found in these two complementary peptides. By comparison with larger fragments, we were able to align the constituent peptides of ankyrin and propose a low resolution model. Ankyrin appears to be a bipolar molecule containing a basic domain of 82,000 daltons and a neutral phosphorylated domain of 55,000 daltons.     

Correlation of exons with structural domains in alcohol dehydrogenase.

The intron/exon arrangement in the gene sequence of maize alcohol dehydrogenase has been compared to the three dimensional structure of liver alcohol dehydrogenase. The co-enzyme binding domain is separated from the catalytic domain by introns four and nine. Intron seven separates the co-enzyme binding domain into two structurally similar mononucleotide binding units. The first of these units is divided by introns five and six into three structurally similar alpha beta modules. Implications of these results for protein evolution is discussed. All splice junctions map close to or at the surface of the domains, and several of these cannot be identified by distance maps.     

Characterization of tryptic hydrolysis of alpha-lactalbumin/saponin mixture and structural change of alpha-lactalbumin interacting with soybean saponin

Bovine milk alpha-lactalbumin (alpha-La) was mixed with soybean saponin, and the resulting mixture was hydrolyzed by trypsin. Saponin increased the tryptic-hydrolysis level of alpha-La only at relatively high phosphate buffer concentrations (> or = 0.05 M). T(1) experiments with acetylated soybean saponin demonstrated that there were some interactions between alpha-La and saponin not only at high concentrations of phosphate buffers but even at low concentrations as well. Circular dichroism spectra of alpha-La showed that the tertiary structure of alpha-La was changed through interactions with saponin only at high buffer concentrations. Furthermore, by analyzing the tryptic peptides from an alpha-La/saponin mixture, hydrolyzing rates at all or some of K5, R10, and K16 of alpha-La were accelerated by saponin interactions. The increase in the tryptic hydrolysis of alpha-La by saponin addition was considered due to modification of the tertiary structure of alpha-La by saponin.     

Dynamic regimes and correlated structural dynamics in native and denatured alpha-lactalbumin

Understanding the mechanisms of protein folding requires knowledge of both the energy landscape and the structural dynamics of a protein. We report a neutron-scattering study of the nanosecond and picosecond dynamics of native and the denatured alpha-lactalbumin. The quasielastic scattering intensity shows that there are alpha-helical structure and tertiary-like side-chain interactions fluctuating on sub-nanosecond time-scales under extremely denaturing conditions and even in the absence of disulfide bonds. Based on the length-scale dependence of the decay rate of the measured correlation functions, the nanosecond dynamics of the native and the variously denatured proteins have three dynamic regimes. When 0.051.0 A(-1) is a regime that displays the local dynamic behavior of individual residues, Gamma proportional to Q(1.8+/-0.3). The picosecond time-scale dynamics shows that the potential barrier to side-chain proton jump motion is reduced in the molten globule and in the denatured proteins when compared to that of the native protein. Our results provide a dynamic view of the native-like topology established in the early stages of protein folding.     

Energetic basis of structural stability in the molten globule state: alpha-lactalbumin

The denatured states of alpha-lactalbumin, which have features of a molten globule state, have been studied to elucidate the energetics of the molten globule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity increment of alpha-lactalbumin denaturation highly correlates with the degree of disorder of the residual structure of the state. As a result, the denaturational transition of alpha-lactalbumin from the native to a highly ordered compact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entropy of the denaturation of alpha-lactalbumin to compact denatured state are always greater than the enthalpy and entropy of its unfolding. This difference represents the unfolding of the molten globule state. Calorimetric measurements of the heat effect associated with the unfolding of the molten globule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic specificity of the molten globule state, which, in contrast to a protein with unique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and enthalpy of internal interactions. It is concluded that at physiological temperatures the entropy of dehydration is the dominant factor providing stability for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor.     

Identification of structural domains in proteins by a graph heuristic.

A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward.     

Membrane structural domains. Resolution limits using diphenylhexatriene fluorescence decay.

Measurement of multiple fluorescence decay times of 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes can in principle be used to investigate structural domains of lipid bilayers. To assess the feasibility of this approach using phase and modulation techniques, we reduced experimental errors specifically associated with performing these measurements on membrane suspensions (probe self-quenching, background fluorescence, turbidity-induced artifacts) and determined empirically the level of precision thereby obtainable. Next we used these precision limits in theoretical calculations to conclude that the ratio of two coexisting decay times must exceed 1.3 if they are to be resolved with reliable accuracy. To demonstrate that such resolutions could be accomplished experimentally in membrane suspensions, three approaches were taken. First, the fluorescence decay of aqueous quinine sulfate quenched by chloride ion was resolved from that of membrane-associated DPH as long as the lifetime ratios of these two fluorophores exceeded the predicted value. Second, populations of DPH-containing lipid vesicles with single (or nearly single) decay times were mixed together, and when there were only two major lifetime components that differed by more than 30%, the resulting heterogeneous fluorescence could be resolved into the two expected lifetime components. Finally, DPH fluorescence decay measurements were correlated with phase behavior in well-characterized lipid systems, revealing a short lifetime component of DPH fluorescence associated with gel-phase lipid vesicles. From these studies, we conclude that only in special cases can co-existing gel and fluid phases be resolved by means of DPH lifetime heterogeneity, within the limits of precision defined herein.     

Conserved structural determinants in three-fingered protein domains

The three-dimensional structures of some components of snake venoms forming so-called 'three-fingered protein' domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-beta receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C-terminal part of TFPDs are essential for the TFPD-like fold. Analyses of the three-dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD-like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.     

Primary structure of rabbit alpha-lactalbumin.

Rabbit alpha-lactalbumin was purified from the milk of New Zealand White rabbits. It was found to exist predominantly as a glycoprotein, containing 5 mol of glucosamine per mol of protein, as well as other sugars. The amino acid sequence of the protein was determined by sequenator analysis and carboxypeptidase digestion. There are 122 amino acids in the protein and a single carbohydrate moiety, probably attached to an asparagine residue at position 45. The C terminus of rabbit alpha-lactalbumin is one residue shorter than that of the other alpha-lactalbumins. Sequence comparisons indicate that the alpha-lactalbumin gene has been undergoing more frequent mutation than had previously been thought. A new method of preparative peptide mapping using 2,5-diphenyloxazole (PPO) fluor to detect peptides is described.