RecA protein self-assembly. Multiple discrete aggregation states

Light scattering, sedimentation and electron microscopy have been used to investigate the aggregation states of highly purified RecA protein in solution. We show that RecA protein will self-assemble into a discrete series of quaternary structures depending upon protein concentration, ionic environment, and nucleotide cofactors. In a stock solution at moderate concentration (10 to 50 microM) RecA protein exists as small particles approximately 4 nm in diameter, larger particles approximately 12 nm in diameter (most probably rings of RecA protein), 10 nm diameter rods varying from 50 to 200 nm in length, and finally as much larger bundles of rods. The addition of monovalent salt shifts the distribution of RecA protein between its various oligomeric states. Increasing protein concentration favors more highly aggregated structures. At a given protein concentration, addition of mM levels of MgCl2 promotes the rapid formation of rods and slow formation of bundles. Under conditions typical of in vitro strand exchange reactions, RecA protein was found to exist as a mixture of rods and 12 nm particles with relatively few monomers.     

Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES.

We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperonin GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin. The refolding reactions induced by stopped-flow pH jumps were monitored by alpha-lactalbumin tryptophan fluorescence. The simple single-exponential character of the free-refolding kinetics of the protein allowed us to quantitatively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for binding between GroEL and the refolding intermediate of alpha-lactalbumin by the non-linear least-squares method. When GroES was absent, the interaction between GroEL and alpha-lactalbumin could be well represented by a "cooperative-binding" model in which GroEL has two binding sites for alpha-lactalbumin with the affinity of the second site being tenfold weaker than that of the first, so that there is negative cooperativity between the two sites. The affinity between GroEL and alpha-lactalbumin was significantly reduced when ATP was present, while ADP and AMP-PNP did not alter the affinity. A comparison of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to the nucleotide-induced reduction of affinity. When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between GroEL and the refolding intermediate of alpha-lactalbumin. The affinity at a saturating concentration of ADP or AMP-PNP was about ten times lower than with GroEL alone. The ADP concentration at which the acceleration of the GroEL/ES-affected refolding of alphaLA was observed, was higher than the concentration at which the nucleotide-induced formation of the GroEL/ES complex took place. These results indicate that GroEL/ES complex formation itself is not enough to reduce the affinity for alpha-lactalbumin, and that further binding of the nucleotide to the GroEL/ES complex is required to reduce the affinity.     

Determination of microvessel permeability and tissue diffusion coefficient of solutes by laser scanning confocal microscopy

Interstitium contains a matrix of fibrous molecules that creates considerable resistance to water and solutes in series with the microvessel wall. On the basis of our preliminary studies, by using laser-scanning confocal microscopy and a theoretical model for interstitial transport, we determined both microvessel solute permeability (P) and solute tissue diffusion coefficient (D) of alpha-lactalbumin (Stokes radius 2.01 nm) from the rate of tissue solute accumulation and the radial concentration gradient around individually perfused microvessel in frog mesentery. P(alpha-lactalbumin) is 1.7 +/- 0.7(SD) x 10(-6) cm/s (n = 6). D(t)/D(free) for alpha-lactalbumin is 27% +/- 5% (SD) (n = 6). This value of D(t)/D(free) is comparable to that for small solute sodium fluorescein (Stokes radius 0.45 nm), while p(alpha-lactalbumin) is only 3.4% of p(sodium fluorescein). Our results suggest that frog mesenteric tissue is much less selective to solutes than the microvessel wall.     

Stability of HAMLET--a kinetically trapped alpha-lactalbumin oleic acid complex

The stability toward thermal and urea denaturation was measured for HAMLET (human alpha-lactalbumin made lethal to tumor cells) and alpha-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than alpha-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15 degrees C lower for apo HAMLET than for apo alpha-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.     

Label-free detection of cupric ions and histidine-tagged proteins using single poly(pyrrole)-NTA chelator conducting polymer nanotube chemiresistive sensor

Novel chemical and biological sensors based on a single poly(pyrrole)-NTA chelator nanotube for sensitive, selective, rapid and real-time detection of histidine-tagged protein and cupric ions are reported. NTA groups on the nanotube surface provided a simple mechanism for metal ion sensing via the high-affinity interaction between NTA and the subsequent detection of histidine-tagged protein through the coordination with metal chelated nanotube. Poly(pyrrole)-NTA chelator nanotubes of 190 nm outside diameter, 35 nm wall thickness and 30 microm long were synthesized by electrochemical polymerization of pyrrole-NTA inside a 200 nm diameter alumina template and assembled as a chemoresistive device by bottom-up contact geometry on a pair of parallel gold electrodes with a gap distance of 3 microm. The chemoresistive sensors based on single poly(pyrrole)-NTA chelator nanotube exhibited detection as low as one-hundredth attomolar (0.6 ppt) cupric ions and 1 ng/ml of penta-histidine tagged syntaxin protein.     

Self Assembly of Short Aromatic Peptides into Amyloid Fibrils and Related Nanostructures

The formation of amyloid fibrils is the hallmark of more than twenty human disorders of unrelated etiology. In all these cases, ordered fibrillar protein assemblies with a diameter of 7-10 nm are being observed. In spite of the great clinical important of amyloid-associated diseases, the molecular recognition and self-assembly processes that lead to the formation of the fibrils are not fully understood. One direction to decipher the mechanism of amyloid formation is the use of short peptides fragments as model systems. Short peptide fragments, as short as pentapeptides, were shown to form typical amyloid assemblies in vitro that have ultrastructural, biophysical, and cytotoxic properties, as those of assemblies that are being formed by full length polypeptides. When we analyzed such short fragments, we identified the central role of aromatic moieties in the ability to aggregate into ordered nano-fibrillar structures. This notion allowed us to discover additional very short amyloidogenic peptides as well as other aromatic peptide motifs, which can form various assemblies at the nano-scale (including nanotubes, nanospheres, and macroscopic hydrogels with nano-scale order). Other practical utilization of this concept, together with novel β-breakage methods, is their use for the development of novel classes of amyloid formation inhibitors.     

Thermodynamics of alpha-lactalbumin-DL-alpha-dipalmitoylphosphatidylcholine interactions and effect of the antioxidant nicotinamide on these interactions

Differential scanning calorimetry has been used to understand the thermodynamics of the interactions of dl-alpha-dipalmitoylphosphatidylcholine (DPPC) with alpha-lactalbumin and the effect of the antioxidant nicotinamide on these interactions. Nicotinamide decreases the thermal transition temperature of both the lipid and the protein at high concentrations. The thermal unfolding transitions of the protein were two state and calorimetrically reversible. There was no significant change in the shape and thermodynamic parameters accompanying the lipid endotherms, suggesting that nicotinamide did not penetrate the lipid bilayer. The thermal unfoldings of alpha-lactalbumin in the presence of DPPC as cosolute also adhered to two-state reversible mechanism. The changes in the thermodynamic parameters accompanying the thermal transitions were small, indicating no significant interaction of alpha-lactalbumin with DPPC. The changes in the thermodynamic parameters indicate that the lipid bilayer organization, as well as the partitioning of the extrinsic protein alpha-lactalbumin into the bilayer, is not affected in the entire studied concentration range of the lipid. It is observed that the presence of increasing concentration of nicotinamide (as high as 1.0 mol dm(-3)) in the lipid-protein mixture does not affect its partitioning into the lipid bilayer, although nicotinamide preferentially interacts with alpha-lactalbumin. The change in the effect of nicotinamide on lipid transition temperature in the mixture and literature report suggests that nicotinamide may be forming a hydrogen-bonded complex with the protein through its amide functionality. The surface tension data of aqueous nicotinamide in combination with the thermal denaturation results of protein in presence of nicotinamide confirmed that surface tension effect does not have any significant contribution to the effect of nicotinamide on protein.     

Self Assembly of Short Aromatic Peptides into Amyloid Fibrils and Related Nanostructures

The formation of amyloid fibrils is the hallmark of more than twenty human disorders of unrelated etiology. In all these cases, ordered fibrillar protein assemblies with a diameter of 7–10 nm are being observed. In spite of the great clinical important of amyloidassociated diseases, the molecular recognition and self-assembly processes that lead to the formation of the fibrils are not fully understood. One direction to decipher the mechanism of amyloid formation is the use of short peptides fragments as model systems. Short peptide fragments, as short as pentapeptides, were shown to form typical amyloid assemblies in vitro that have ultrastructural, biophysical, and cytotoxic properties, as those of assemblies that are being formed by full length polypeptides. When we analyzed such short fragments, we identified the central role of aromatic moieties in the ability to aggregate into ordered nano-fibrillar structures. This notion allowed us to discover additional very short amyloidogenic peptides as well as other aromatic peptide motifs, which can form various assemblies at the nano-scale (including nanotubes, nanospheres, and macroscopic hydrogels with nano-scale order). Other practical utilization of this concept, together with novel β breakage methods, is their use for the development of novel classes of amyloid formation inhibitors.Key Words: Alzheimer''s disease, amyloid disease, molecular recognition, nanostructures, protein aggregation, protein misfolding, self-assembly, type II diabetes     

Kinetics of disulfide bond reduction in alpha-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond

Kinetics of disulfide reduction in alpha-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25 degrees C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and -120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by bimolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide alpha-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon alpha-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.     

Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin

Comparative studies of the unfolding equilibria of two homologous proteins, bovine alpha-lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of alpha-lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein. In previous studies, alpha-lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of alpha-lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, alpha-lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the featureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+, the unfolding transition of alpha-lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native alpha-lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.     

A study of strontium binding to albumins, by a chromatographic method involving atomic emission spectrometric detection

A chromatographic method involving ICP-AES (inductively coupled plasma atomic emission spectrometry) detection has been successfully applied for the study of strontium-protein complexes. The chromatographic step involves the use of gel filtration-a large-zone Hummel and Dreyer method-which allows to dissociate the bound metallic ions and the free ones. This step is followed by an ICP-AES analysis of fractions collected throughout the chromatographic experiment: the concentration of ionic metallic species in solution can therefore be calculated. Two proteins have been tested: bovine serum albumin, which showed only weak interactions with Sr2+ ions, and bovine alpha-lactalbumin: this protein, well-known for its calcium binding capacity, proved to interact strongly with strontium. The influence of various parameters on the formation of strontium-lactalbumin complexes were determined, namely temperature, pH. Competition experiments between Sr2+ ions and, respectively Na+ and Ca2+ ions were also performed, by varying ionic strength of the medium, and by using both apo and native forms of bovine alpha-lactalbumin.     

The mode of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin.

Molecular chaperones prevent the aggregation of partially folded or misfolded forms of protein. alpha-crystallin performs such a function in the ocular lens. To gain insight into the mechanism of the anti-aggregation activity of alpha-crystallin, we performed dynamic light scattering (DLS) measurements investigating its interaction with partially denatured alpha-lactalbumin over a 24 hr period. Analyses were conducted as a function of the concentration of alpha-lactalbumin as well as the bovine alpha-crystallin/alpha-lactalbumin ratio. Additional studies of the systems were performed by HPLC and SDS gel electrophoresis. The particle distribution patterns derived from the DLS data indicated that the chaperoned complex (lactalbumin plus crystallin) is a loose fluffy globular entity. After the complex becomes saturated with lactalbumin, it appears to release the partially denatured lactalbumin which may aggregate into high molecular weight moieties. These eventually may precipitate out of solution. On longer standing, 24hr and over, the chaperoned complex as well as the lactalbumin aggregates become more compact. The chaperoned complex (alpha-crystallin plus alpha-lactalbumin) is in dynamic equilibrium both with the monomeric and the aggregated alpha-lactalbumin population.     

Kinetics of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin

Molecular chaperones prevent the aggregation of partially folded or misfolded forms of protein. alpha-Crystallin performs such a function in the ocular lens. Dynamic light scattering (DLS) measurements were performed to gain insight into the kinetics and mechanism of alpha-crystallin chaperoning. Experiments were conducted as a function of alpha-lactalbumin concentration as well as the alpha-crystallin/alpha-lactalbumin ratio over a 24 h period. In the particle distribution patterns the lactalbumin concentration was partitioned into three compartments: (a) monomeric free lactalbumin; (b) lactalbumin in the chaperoning complex; and (c) lactalbumin aggregates. DLS intensities were converted to molar concentrations by assuming a model of a spherical chaperoning complex. In the model, alpha-crystallin is the central core and alpha-lactalbumin molecules occupy a ring surrounding the core. The kinetics of chaperoning was studied by proposing a simple scheme with four rate constants. The reversible reaction of the formation of the chaperoning complex is characterized by rate constants k(1) and k(2). The rate constants k(3) and k(4) govern the irreversible aggregation of lactalbumin: the former from the free monomeric lactalbumin pool and the latter describing the aggregation of the denatured lactalbumin released from the chaperoning complex. The rate constants, k(3) and k(4) are four magnitudes larger than k(1) and k(2). The equilibrium constant of chaperoning complex formation lies in favor of the reactants. k(4) is somewhat faster than k(3) and it is three times faster than k(s) governing the self-aggregation of lactalbumin in the absence of alpha-crystallin.     

Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges

Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence spectrum; and (2) a component with kynurenine-like fluorescent properties. The first component has from 0.6 to 0.9 free DTNB-reactive SH groups per protein molecule, which are absent in the native protein and is characterized by slightly lowered Ca(2+)-affinity (2 x 10(8) M(-1) versus 8 x 10(8) M(-1) for the native protein) and absence of observable thermal transition. The second component corresponds to the protein with photochemically modified tryptophan residues. It is assumed that the UV excitation of tryptophan residue(s) in alpha-lactalbumin is followed by a transfer of electrons to the Sbond;S bonds, resulting in their reduction. Mass spectrometry data obtained for trypsin-fragmented UV-illuminated alpha-lactalbumin with acrylodan-modified free thiol groups reveal the reduction of the 61-77 and 73-91 disulfide bridges. The effect observed has to be taken into account in any UV-region spectral studies of alpha-lactalbumin.     

Cooperative folding of the isolated alpha-helical domain of hen egg-white lysozyme.

Proteins in the alpha-lactalbumin and c-type lysozyme family have been studied extensively as model systems in protein folding. Early formation of the alpha-helical domain is observed in both alpha-lactalbumin and c-type lysozyme; however, the details of the kinetic folding pathways are significantly different. The major folding intermediate of hen egg-white lysozyme has a cooperatively formed tertiary structure, whereas the intermediate of alpha-lactalbumin exhibits the characteristics of a molten globule. In this study, we have designed and constructed an isolated alpha-helical domain of hen egg-white lysozyme, called Lyso-alpha, as a model of the lysozyme folding intermediate that is stable at equilibrium. Disulfide-exchange studies show that under native conditions, the cysteine residues in Lyso-alpha prefer to form the same set of disulfide bonds as in the alpha-helical domain of full-length lysozyme. Under denaturing conditions, formation of the nearest-neighbor disulfide bonds is strongly preferred. In contrast to the isolated alpha-helical domain of alpha-lactalbumin, Lyso-alpha with two native disulfide bonds exhibits a well-defined tertiary structure, as indicated by cooperative thermal unfolding and a well-dispersed NMR spectrum. Thus, the determinants for formation of the cooperative side-chain interactions are located mainly in the alpha-helical domain. Our studies suggest that the difference in kinetic folding pathways between alpha-lactalbumin and lysozyme can be explained by the difference in packing density between secondary structural elements and support the hypothesis that the structured regions in a protein folding intermediate may correspond to regions that can fold independently.     

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.     

On the equilibrium between monomeric alpha-lactalbumin and the chaperoning complex of alpha-crystallin

In chaperoning dithiothreitol-denatured alpha-lactabumin, alpha-crystallin forms a chaperoning complex. In order to study the kinetics of such chaperoning it needs to be established whether the formation of the chaperoning complex is a reversible or irreversible process. The chaperoning reaction was studied by dynamic light scattering as a function of concentration and weight ratio of alpha-lactalbumin/alpha-crystallin. HPLC and subsequent SDS-PAGE gel electrophoresis experiments established that the chaperoning complex formed contains both alpha-crystallin and alpha-lactalbumin. Upon rechromatographing the chaperoning complex, the presence of monomeric alpha-lactalbumin has been demonstrated in addition to the chaperoning complex itself. This and equilibrium dialysis experiments demonstrated conclusively the existence of an equilibrium between monomeric partially denatured alpha-lactalbumin and the chaperoning complex made of alpha-lactalbumin and alpha-crystallin.     

Effects of a helix substitution on the folding mechanism of bovine alpha-lactalbumin

The structure, stability, and unfolding-refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105-110) comes from equine lysozyme and the remainder of the sequence comes from bovine alpha-lactalbumin were studied by circular dichroism spectroscopy and stopped-flow measurements, and the results were compared with those of bovine alpha-lactalbumin. The substitution of the flexible loop in bovine alpha-lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine alpha-lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine alpha-lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non-native interaction in the flexible loop region and that the disruption of the non-native interaction reduces the free energy barrier of refolding. We conclude that the non-native interaction in the molten globule state may act as a kinetic trap for the folding of alpha-lactalbumin.     

Protein interactions within the N-end rule ubiquitin ligation pathway

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm). In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.     

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.