Observations on the binding of lanthanides and calcium to vitamin D-dependent chick intestinal calcium-binding protein. Implications regarding calcium-binding protein function

The binding of calcium and terbium to purified chick vitamin D-dependent intestinal calcium-binding protein was studied by terbium fluorescence, circular dichroism, and intrinsic protein fluorescence techniques. Calcium-binding protein bound, with high affinity, at least 3 mol of terbium/mol of protein; numerous low affinity terbium-binding sites were also noted. The three highest affinity sites were resolved into one very high affinity site (site A) and two other sites (sites B and C) with slightly lower affinity. Resonance energy transfer from tryptophan residues to terbium occurred only with site A. This site was filled before sites B and C. Competition experiments in which calcium was used to displace terbium bound to the protein showed that larger amounts of calcium were needed to displace terbium from site A than from sites B and C. Energy transfer from terbium to holmium indicated that the terbium-binding sites (B and C) were located close to each other (about 7-12 A) but were distant (greater than 12 A) from site A. The addition of EDTA to calcium-binding protein resulted in a 25% decrease in intrinsic protein fluorescence, suggesting a conformational change in the protein. The titration of EDTA-treated calcium-binding protein with calcium resulted in recovery of intrinsic protein fluorescence. A reversible calcium-dependent change in the ellipticity of calcium-binding protein in circular dichroism experiments was also seen. These observed properties suggest that vitamin D-dependent chick intestinal calcium-binding protein behaves in a manner similar to other well-known calcium-binding regulatory proteins.     

Chemical modification of prothrombin fragment 1: documentation of sequential, two-stage loss of protein function

The amino groups of prothrombin fragment 1 (amino acids 1-156 of prothrombin) were derivatized by acetylation, amidination, and reductive methylation. Conditions that caused complete acetylation of protein amino groups produced a fragment 1 derivative which no longer displayed a metal ion dependent intrinsic fluorescence change and had lost its membrane binding capability as well. However, when derivatized in the presence of calcium ions, extensive acetylation yielded a product that underwent protein fluorescence quenching at metal ion concentrations similar to those observed for the native protein. This derivative bound to membranes in a calcium-dependent manner with only a small reduction in affinity. Several results showed the existence of a partially functional protein that was characterized by a high degree of calcium-dependent protein fluorescence quenching but which had a requirement for 10-fold higher calcium concentration. This derivative was produced by partial acetylation (greater than 3 equiv) of metal-free protein. This partially acetylated protein had greatly diminished membrane binding. The calcium-protected amino group, therefore, was among the most reactive acetylation sites in the metal-free protein. The second site, responsible for abolishing all metal ion induced fluorescence change, was resistant to acetylation and became derivatized at the last stages of amino group acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of oligosaccharides and chloride on the oligomeric structures of external, internal, and deglycosylated invertase

External invertase exists in an oligomeric equilibrium of dimer, tetramer, hexamer, and octamer, the concentrations of which vary with pH, time, and concentration of enzyme [Chu, F.K., Watorek, W., & Maley, F. (1983) Arch. Biochem. Biophys. 223, 543-555; Tammi, M., Ballou, L., Taylor, A., & Ballou, C.E. (1987) J. Biol. Chem. 262, 4395-4401]. To assess the influence of carbohydrate on this equilibrium, we investigated the self-association of external invertase (10 oligosaccharides per subunit), deglycosylated external invertase (2 oligosaccharides per subunit), and internal invertase (no carbohydrate) under various conditions. In addition, the effect of carbohydrate on the interaction of the subunits of these various invertases to form heterooligomers was studied. Chloride ion was found to promote subunit association in the various invertases irrespective of their glycosylation status. However, external invertase was less responsive to chloride ion relative to the internal and deglycosylated invertases. The higher oligomers of deglycosylated invertase were stable at 47 degrees C whereas those of external invertase dissociated rapidly into dimers, suggesting that the additional oligosaccharides in external invertase destabilize subunit interaction. Hybridization experiments with the various invertases showed that the dimers of internal invertase formed heterooligomers with either external or deglycosylated invertase. By contrast, the monomers of external and internal invertases formed their respective homodimers, but not heterodimers. These results suggest that the oligosaccharide content of invertase not only influences the extent of self-association but also affects heterooligomer formation.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase

To study the influence of N-linked carbohydrate moiety on the catalytic and biochemical properties of glycosylated enzyme, a recombinant β-d-glucuronidase (PGUS-P) from Penicillium purpurogenum as a model glycoprotein, was deglycosylated with peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic deglycosylation procedure resulted in the complete removal of carbohydrate moiety. Compared with the glycosylated PGUS-P, the deglycosylated PGUS-P exhibited 20-70% higher activity (p<0.05) within pH 6-9, but 15-45% lower activity (p<0.05) at 45-70°C. The apparent decrease in the thermal stability of the deglycosylated enzyme was reflected by a decrease in the denaturation temperature (T(d)) values determined by differential scanning calorimetry (DSC). The removal of N-linked glycans also reduced enzyme's sensitivity to certain metal ions. The deglycosylated PGUS-P displayed lower K(m) vaules, but higher k(cat)/K(m) ratios than the glycosylated isoform towards glycyrrhizin. The consequent conformational changes were also determined by circular dichroism (CD) and fluorescence spectroscopy which revealed no significant difference in the secondary but a slight dissimilarity between the tertiary structures of both isoforms of PGUS-P.     

Synthesis and conformational features of human salivary mucin C-terminal derived peptide epitope carrying Thomsen-Friedenreich antigen: implications for its role in self-association

The conformational features of a chemically synthesized 23-residue glycopeptide construct (II) carrying Gal-beta-(1,3)-alpha-GalNAc and its deglycosylated counterpart (I; Gal: galactose; GalNAc: N-acetyl galactosamine) derived from the C-terminal domain of human salivary mucin (MUC7) were investigated using CD spectroscopy as well as molecular dynamic simulation studies. The corresponding deglycosylated peptide (I) was essentially used to compare and study the influence of the sugar moiety on peptide backbone conformation. CD measurements in aqueous medium revealed that the apopeptide (I) contains significant populations of beta-strand conformation while the glycopeptide (II) possess, partly, helical structure. This transition in the secondary structure upon glycosylation from beta-strand to helical conformation clearly demonstrates that the carbohydrate moiety exerts significant influence on the peptide backbone. On the other hand, upon titrating structure stabilizing organic cosolvent, trifluoroethanol (TFE), both the peptides showed pronounced helical structure. However, the propensity for helical structure formation is less pronounced in glycopeptide compared to apopeptide suggesting that the bulky carbohydrate moiety possibly posing steric hindrance to the formation of TFE-induced secondary structure in II. Energy-minimized molecular model for the glycopeptide revealed that the preferred helix conformation in aqueous medium appears to be stabilized by the hydrogen-bonded salt bridge like interaction between carbohydrate --OH and Lys-10 side--N(+)H(3) group. Size exclusion chromatographic analysis of both (glyco)peptides I and II showed an apparent Kd of 2.3 and 0.52 microM, respectively, indicating that glycopeptide (II) has greater tendency for self-association. Due to high amphipathic character as well as due to the presence of a leucine zipper motif ( approximately LLYMKNLL approximately ), which is known to increase the stability at the coiled-coil interface via hydrophobic interactions, we propose therefore that, this domain could be one of the key elements involved in the self-association of intact MUC7 in vivo. Profound conformational effects governed by glycosylation exemplified herein could have implications in determining structure-function relationships of mucin glycoproteins.     

Ligand induced galectin-3 protein self-association

Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in cross-linking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here we studied interaction of galectin-3 with the model glycoprotein asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concentration of free galectin carbohydrate recognition sites in solution. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concentrations, showing that many more galectin-3 molecules were engaged than expected due to the about nine known glycan-based binding sites per ASF molecule. This suggests that after ASF-induced nucleation, galectin-3 associates with itself by the carbohydrate recognition site binding to another galectin-3 molecule, possibly forming oligomers. We named this type-C self-association to distinguish it from the previously proposed models (type-N) where galectin-3 molecules bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-association can result in precipitates, as measured here by turbidimetry and dynamic light scattering. Type-C self-association and precipitation occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required much higher concentration (～50 μM) as compared with wild type (～1 μM). ASF also induced weaker type-C self-association of galectin-3 lacking its N-terminal domains, but as expected, no precipitation. Neither a monovalent nor a divalent N-acetyl-D-lactosamine-containing glycan induced type-C self-association, even if the latter gave precipitates with high concentrations of galectin-3 (>～50 μM) in agreement with published results and perhaps due to type-N self-association.     

Human RegIV Protein Adopts a Typical C-Type Lectin Fold but Binds Mannan with Two Calcium-Independent Sites

Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of ¹⁵N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract.     

Glycosylation of purified buffalo heart galectin-1 plays crucial role in maintaining its structural and functional integrity

A buffalo heart galectin-1 purified by gel filtration chromatography revealed the presence of 3.55% carbohydrate content, thus it is the first mammalian heart galectin found to be glycosylated in nature and emphasizes the need to perform deglycosylation studies. Physicochemical comparative analysis between the properties of the native and deglycosylated proteins was carried out to understand the significance of glycosylation. The deglycosylated protein exhibited lesser thermal and pH stability compared to the native galectin. When exposed to thiol blocking reagents, denaturants, and detergents, remarkable differences were observed in the properties of the native and deglycosylated protein. Compared to the native glycosylated protein, the deglycosylated galectin showed enhanced fluorescence quenching when exposed to various agents. CD and FTIR analysis showed that deglycosylation of the purified galectin and its exposure to different chemicals resulted in significant deviations from regular secondary structure of the protein, thus emphasizing the significance of glycosylation for maintaining the active conformation of the protein. The remarkable differences observed in the properties of the native and deglycosylated galectin add an important dimension to the significance of protein glycosylation and its associated biological and clinical relevance.     

Enzymatic deglycosylation of human thyroglobulin: fluorescence studies

The interaction between the carbohydrate and the amino acid residues in human thyroglobulin has been studied. Previous reports showed that the removal of the two terminal carbohydrates of the complex chains leads to an increase in thyroglobulin binding to thyroid membranes. In our study, after enzymatic release with glycosidases of the sugar moieties from thyroglobulin, a time-dependent decrease in tryptophan fluorescence has been observed. This decrease was also associated with a shift in the emission peak from 335 to 340 nm. The strong quenching of tryptophan emission was also accompanied by a decrease in the exposure of tryptophan residues, as shown by a Stern-Volmer analysis with the neutral quencher acrylamide. These data, together with the increase in fluorescence of the dansylated deglycosylated thyroglobulin, strongly suggest that a significant conformational change of thyroglobulin follows the deglycosylation of the protein.     

Role of the carbohydrate part of yeast acid phosphatase

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.     

Role of carbohydrate in human chorionic gonadotropin: Deglycosylation uncouples hormone-receptor complex and adenylate cyclase system

Previous work has shown that deglycosylation of human chorionic gonadotropin (hCG) does not affect its receptor binding characteristics, but its ability to stimulate intracellular cyclic AMP accumulation and steroidogenesis in ovarian cells is abolished. To identify the site at which carbohydrate of hCG is involved in the mechanism of action of the hormone, we have studied adenylate cyclase activity in ovarian membrane preparations in response to deglycosylated and native hCG. The deglycosylated hCG does not stimulate adenylate cyclase of ovarian membrane preparation and also it acts as an inhibitor of hCG action. Data are presented to show that both hCG- and catecholamine receptors are coupled to the same adenylate cyclase complex. Since adenylate cyclase activity in the presence of deglycosylated hCG remains still responsive maximally to catecholamines, it indicates that the adenylate cyclase complex is functional and is unaffected by the interaction of deglycosylated hCG to its receptor. This is further supported by the fact that the deglycosylated hCG does not impair the maximal stimulation of adenylate cyclase by guanine nucleotides. Thus, the site of action of the carbohydrate of hCG is prior to the coupling of the hormone-receptor complex and the adenylate cyclase system.     

Mechanism of photoinhibition in vivo. A reversible light-induced conformational change of reaction center II is related to an irreversible modification of the D1 protein

The light-induced inactivation of the photochemical reaction center II (RCII) of oxygenic chloroplasts (photoinhibition) was investigated in cells and isolated thylakoids of the green alga Chlamydomonas reinhardtii. The process is resolved into a reversible conformational change followed by an irreversible modification of RCII D1 protein. The light-induced changes in vivo persisted in isolated thylakoids. The first step is characterized by (i) destabilization of the secondary acceptor semiquinone anion, Q-B, bound to the D1 protein. This is demonstrated by a reduction in the activation energy of S2,3Q-B charge recombination as measured by the thermoluminescence technique; and (ii) a rise in the intrinsic fluorescence and a decrease of the maximal fluorescence. Unoccupancy of the QB site by plastoquinone partially protected RCII against the light-induced destabilization of Q-B. The extent of charge separation (P+680Q-A) was not affected. However, the slow phase (microsecond) of P+680 dark reduction increased, and the amplitude of signal II was reduced by 20-30%, indicating that in a fraction of RCII, electron donation from Z to P+680 was impaired without losing primary photochemistry. This modification correlates with the irreversible change in D1 protein resulting in the formation of a trypsin-resistant fragment of 16 kDa detected in D1 isolated from light-exposed cells. The change in the Q-B stability could allow charge equilibration with QA and thus explain the rise in the intrinsic fluorescence level and reduction of electron flow to plastoquinone. The change in the lifetime of P+680 can account for further reduction in electron flow (photo-inhibition). The irreversible light-dependent modification of D1 may serve as the signal for its degradation and replacement by a newly synthesized molecule (turnover).     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified (Na+ + K+)-ATPase accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to beta-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of (Na+ + K+)-ATPase and p-nitrophenylphosphate activity by digitonin (which occurs during the rapid phase of inactivation) is unlikey to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either (Na+ + K+)-ATPase or p-nitrophenylphosphatase activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants

We have substituted leucine 56 or tyrosine 64 of p21 ras with a tryptophan. The intrinsic fluorescence of this tryptophan was used as an internal conformational probe for time-resolved biochemical studies of the ras protein. The slow intrinsic GTPase, GDP/GTP exchange induced by the SDC25 "exchange factor", and the fast GTP hydrolysis induced by GAP were studied. Tryptophan fluorescence of mutated ras is very sensitive to magnesium binding, GDP/GTP exchange, and GTP hydrolysis (changes in tyrosine fluorescence of wild-type ras are also observed but with a lower sensitivity). Nucleotide affinities, exchange kinetics, and intrinsic GTPase rates of the mutated ras could be measured by this method and were found to be close to those of wild-type ras. The SDC25 gene product enhances GDP/GTP exchange in both mutants. In both mutants, a slow fluorescence change follows the binding of GTP gamma S; its kinetics are close to those of the intrinsic GTPase, suggesting that a slow conformational change precedes the GTPase and is the rate-limiting step, as proposed by Neal et al. (1990) (Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565). GAP interacts with both mutant ras proteins and accelerates the GTPase of (L56W)ras but not that of (Y64W)ras, suggesting a role for tyrosine 64 in GAP-induced GTP hydrolysis. However, GAP does not accelerate the slow conformational change following GTP gamma S binding in either of the mutated ras proteins. This suggests that the fast GAP-induced catalysis of GTP hydrolysis that is observed with (L56W)ras bypasses the slow conformational change associated with the intrinsic GTPase and therefore might proceed by a different mechanism.     

A flagellum-specific calcium sensor

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.     

Denaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride; identification of inactive, partially unfolded, dimeric intermediates.

The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride has been studied using enzymatic activity, intrinsic fluorescence, circular dichroism, and light scattering measurements. Equilibrium experiments at 25 degrees C revealed that between 0.9 and 1.2 M denaturant the enzyme underwent a conformational change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits. This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation, perhaps due to exposure of 'sticky' hydrophobic stretches of the polypeptide chain. A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurred only at denaturant concentrations above 1.4 M. Kinetics experiments demonstrated that in the denaturant concentration range of 1.7-1.9 M the fluorescence change occurred in two distinct steps. The first step involved a large, very rapid drop in fluorescence whose rate was strongly dependent on the denaturant concentration. This was followed by a small, relatively slow rise in the emission intensity, the rate of which was independent of denaturant concentration. Enzymatic activity was lost with a denaturant-concentration-dependent rate, which was approx. 3-times slower than the rate of the first step in fluorescence change. A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed. While the fully unfolded enzyme regained up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates were able to reactivate only minimally and in fact were found to aggregate and precipitate out of solution.     

Three-state unfolding and self-association of maspin, a tumor-suppressing serpin.

Maspin is a tumor suppressor protein expressed by normal human mammary epithelium but not by many breast tumor cell lines. Recombinant human maspin (rMaspin) inhibits tumor cell motility, invasion, and metastasis and thus has potential value as an anti-cancer therapeutic. Maspin is a member of the serpin family and, although the molecular mechanism by which maspin acts is unknown, recent work suggests that tissue plasminogen activator is a potential target. A puzzling observation in previous cell culture studies was loss of rMaspin activity at higher protein concentrations. One hypothesis to explain these results is self-association of rMaspin at the higher concentrations, which would be consistent with the tendency of serpins to form noncovalent polymers. This hypothesis is addressed by examining the relationship between rMaspin stability and self-association. Urea denaturation of rMaspin at pH 7 and 25 degrees C and at protein concentrations ranging from 0.01 to 0.2 mg/ml has been monitored by circular dichroism and intrinsic tryptophan fluorescence. Denaturation profiles show a protein concentration dependence and indicate the presence of at least one unfolding intermediate. The results suggest that destabilization of native monomeric rMaspin leads to partial unfolding and formation of an intermediate which can self-associate.     

Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement in Rubisco activase upon ATP binding

Two species-invariant tryptophan residues at positions 109 and 250 of tobacco Rubisco activase were identified by site-directed mutagenesis as being responsible for the increase in intrinsic fluorescence upon addition of ATP, which has been previously attributed to increased self-association. Substitution of W109, which is immediately prior to a ‘P-loop’ sequence in the ATP catalytic motif, with aromatic residues (Tyr or Phe), Cys or Lys eliminated both ATP hydrolysis and the intrinsic fluorescence enhancement. Although the W109 mutants bound ATP, ATP did not provide a partial protection against proteolysis by trypsin that was observed with the recombinant wild-type enzyme. In contrast, substitution of W250 with Tyr or Phe abolished about half (44%) of the increase in intrinsic fluorescence with ATP, but had little effect on ATP hydrolysis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation or proteolytic protection with ATP. The substitution of the other tryptophan residues, W16 and W305, with phenylalanine did not significantly alter the change in intrinsic fluorescence upon addition of ATP. Therefore, W109 and W250 are the residues reporting the conformational change that increases the intrinsic fluorescence.     

Effects of deglycosylation of sodium channels on their structure and function

Voltage-gated sodium channels are important membrane proteins underlying electrical signaling in the nervous and muscular systems. They undergo rapid conformational changes between closed resting, activated, and inactivated states. Approximately 30% of the mass of the sodium channel is carbohydrate, present as glycoconjugate chains, mostly composed of N-acetylhexosamines and sialic acid. In this study, the effects of removing the carbohydrate on the functional and structural properties of highly purified sodium channels from Electrophorus electricus were investigated. After enzymatic deglycosylation, channels were reconstituted into planar lipid bilayers. In the presence of batrachotoxin, substates became evident and the single-channel conductance of the deglycosylated channels was slightly reduced relative to that of native channels, consistent with electrostatic effects due to the reduction in negative charge at the extracellular vestibule of the channel. The previously reported state-dependent changes in the circular dichroism spectra that are associated with the binding of the anticonvulsant drug Lamotrigine and batrachotoxin are also seen in the modified channels. Synchrotron radiation circular dichroism (SRCD) spectroscopy on the type of sugars found in the sodium channel showed that unlike most carbohydrates, these sugars produce a significant dichroic signal in the far-ultraviolet region. This can account for all of the measured SRCD-detected spectral differences between the native and deglycosylated channels, thereby indicating that no net change in protein secondary structure results from the deglycosylation procedure. Furthermore, thermal denaturation studies detected no significant differences in stability between native and deglycosylated channels. In summary, while the sugars of the voltage-gated sodium channels from electroplax are not essential for functional or structural integrity, they do appear to have a modulating effect on the conductance properties of these channels.