Factors that control the reactivity of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi

The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in TbTIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.     

Crosstalk between the subunits of the homodimeric enzyme triosephosphate isomerase

Homodimeric triosephosphate isomerase (TIM) from Trypanosoma cruzi (TcTIM) and T. brucei (TbTIM) are markedly similar in amino acid sequence and three-dimensional structure. In their dimer interfaces, each monomer has a Cys15 that is surrounded by loop3 of the adjoining subunit. Perturbation of Cys15 by methylmethane thiosulfonate (MMTS) induces abolition of catalysis and structural changes. In the two TIMs, the structural arrangements of their Cys15 are almost identical. Nevertheless, the susceptibility of TcTIM to MMTS is nearly 100-fold higher than in TbTIM. To ascertain the extent to which the characteristics of the interface Cys depend on the dynamics of its own monomer or on those of the adjacent monomer, we studied MMTS action on mutants of TcTIM that had the interface residues of TbTIM, and hybrids that have only one interfacial Cys15 (C15ATcTIM-wild type TbTIM). We found that the solvent exposure of the interfacial Cys depends predominantly on the characteristics of the adjoining monomer. The maximal inhibition of activity induced by perturbation of the sole interface Cys in the C15ATcTIM-TbTIM hybrid is around 60%. Hybrids formed with C15ATcTIM monomers and catalytically inert TbTIM monomers (E168DTbTIM) were also studied. Their activity drops by nearly 50% when the only interfacial Cys is perturbed. These results in conjunction with those on C15ATcTIM-wild type TbTIM hybrid indicate that about half of the activity of each monomer depends on the integrity of each of the two Cys15-loop3 portions of the interface. This could be another reason of why TIM is an obligatory dimer.     

Control of the reactivation kinetics of homodimeric triosephosphate isomerase from unfolded monomers

Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their β‐turn occurrence, we engineered two chimerical enzymes where their super secondary β‐loop‐α motifs 2 ((βα)₂) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (βα)₂ motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (βα)₂ motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (βα)₂ of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N‐terminal of loop‐2 and the C‐terminal of α‐helix‐2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (E_A). A systematic analysis of DSC data showed a large decrease on the E_A of TcTIM (ΔE_A ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571–579. © 2016 Wiley Periodicals, Inc.     

Reversible equilibrium unfolding of triosephosphate isomerase from Trypanosoma cruzi in guanidinium hydrochloride involves stable dimeric and monomeric intermediates

The reversible guanidinium hydrochloride-induced unfolding of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was characterized under equilibrium conditions. The catalytic activity was followed as a native homodimeric functional probe. Circular dichroism, intrinsic fluorescence, and size-exclusion chromatography were used as secondary, tertiary, and quaternary structural probes, respectively. The change in ANS fluorescence intensity with increasing denaturant concentrations was also determined. The results show that two stable intermediates exist in the transition from the homodimeric native enzyme to the unfolded monomers: one (N(2*)) is a slightly more expanded, non-native, and active dimer, and the other is a partially expanded monomer (M) that binds ANS. Spectroscopic and activity data were used to reach a thermodynamic characterization. The results indicate that the Gibbs free energies for the partial reactions are 4.5 (N(2) <==> N(2*)), 65.8 (N(2*) <==> 2M), and 17.8 kJ/mol (M <==> U). It appears that TcTIM monomers are more stable than those found for other TIM species (except yeast TIM), where monomer stability is only marginal. These results are compared with those for the guanidinium hydrochloride-induced denaturation of TIM from different species, where despite the functional and three-dimensional similarities, a remarkable heterogeneity exists in the unfolding pathways.     

Catalysis and stability of triosephosphate isomerase from Trypanosoma brucei with different residues at position 14 of the dimer interface. Characterization of a catalytically competent monomeric enzyme

In homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM), cysteine 14 of each the two subunits forms part of the dimer interface. This residue is central for the catalysis and stability of TbTIM. Cys14 was changed to the other 19 amino acids to determine the characteristics that the residue must have to yield catalytically competent stable enzymes. C14A, C14S, C14P, C14T, and C14V TbTIMs were essentially wild type in activity and stability. Mutants with Asn, Arg, and Gly had low activities and stabilities. The other mutants had less than 1% of the activity of TbTIM. One of the latter enzymes (C14F) was purified to homogeneity. Size exclusion chromatography and equilibrium sedimentation studies showed that C14F TbTIM is a monomer, with a k(cat) approximately 1000 times lower and a K(m) approximately 6 times higher than those of TbTIM. In C14F TbTIM, the ratio of the elimination (methylglyoxal and phosphate formation) to isomerization reactions was higher than in TbTIM. Its secondary structure was very similar to that of TbTIM; however, the quantum yield of its aromatic residues was lower. The analysis of the data with the 19 mutants showed that to yield enzymes similar to the wild type, the residue must have low polarity and a van der Waals volume between 65 and 110 A(3). The results with C14F TbTIM illustrate that the secondary structure of TbTIM can be formed in the absence of intersubunit contacts, and that it has sufficient tertiary structure to support catalysis.     

Inactivation of triosephosphate isomerase from Trypanosoma cruzi by an agent that perturbs its dimer interface

We characterized by crystallographic, calorimetric and biochemical methods the action of a low molecular weight compound, 3-(2-benzothiazolylthio)-1-propanesulfonic acid (compound 8) that binds to the dimer interface of triosephosphate isomerase from Trypanosoma cruzi (TcTIM) and thereby abolishes its function with a high level of selectivity. The kinetics of TcTIM inactivation by the agent and isothermal titration calorimetry experiments showed that the binding of two molecules of the compound per enzyme is needed for inactivation. The binding of the first molecule is endothermic, and that of the second exothermic. Crystals of TcTIM in complex with one molecule of the inactivating agent that diffracted to a resolution of 2A were obtained. The compound is at the dimer interface at less than 4A from residues of the two subunits. Compound 8 is more effective at low than at high protein concentrations, indicating that it perturbs the association between the two TcTIM monomers. Calorimetric and kinetic data of experiments in which TcTIM was added to a solution of the inactivating agent showed that at low concentrations of the compound, inactivation is limited by binding, whereas at high concentrations of the agent, the events that follow binding become rate-limiting. The portion of the interface of TcTIM that binds the benzothiazole derivative and its equivalent region in human TIM differs in amino acid composition and hydrophobic packing. Thus, we show that by focusing on protein-protein interfaces, it is possible to discover low molecular weight compounds that are selective for enzymes from parasites.     

Unfolding of triosephosphate isomerase from Trypanosoma brucei: identification of intermediates and insight into the denaturation pathway using tryptophan mutants

The unfolding of triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) induced by guanidine hydrochloride (GdnHCl) was characterized. In contrast to other TIMs, where unfolding is a two or three state process, TbTIM showed two intermediates. The solvent exposure of different regions of the protein in the unfolding process was characterized spectroscopically with mutant proteins in which tryptophans (W) were changed to phenlylalanines (F). The midpoints of the transitions measured by circular dichroism, intrinsic fluorescence, and catalytic activity, as well as the increase in 1-aniline 8-naphthalene sulfonate fluorescence, show that the native state was destabilized in the W12F and W12F/W193F mutants, relative to the wild-type enzyme. Using the hydrodynamic profile for the unfolding of a monomeric TbTIM mutant (RMM0-1TIM) measured by size-exclusion chromatography as a standard, we determined the association state of these intermediates: D*, a partially expanded dimer, and M*, a partially expanded monomeric intermediate. High-molecular-weight aggregates were also detected. At concentrations over 2.0 M GdnHCl, the hydrodynamic properties of TbTIM and RMM0-1TIM are the same, suggesting that the dimeric intermediate dissociates and the unfolding proceeds through the denaturation of an expanded monomeric intermediate. The analysis of the denaturation process of the TbTIM mutants suggests a sequence for the gradual exposure of W residues: initially the expansion of the native dimer to form D* affects the environments of W12 and W159. The dissociation of D* to M* and further unfolding of M* to U induces the exposure of W170. The role of protein concentration in the formation of intermediates and aggregates is discussed considering the irreversibility of this unfolding process.     

Key residues of loop 3 in the interaction with the interface residue at position 14 in triosephosphate isomerase from Trypanosoma brucei

Cysteine 14 is an interface residue that is fundamental for the catalysis and stability of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM). Its side chain is surrounded by a deep pocket of 11 residues that are part of loop 3 of the adjacent monomer. Mutation of this residue to serine (producing single mutant C14S) yields a wild-type-like enzyme that is resistant to the action of sulfhydryl reagents methylmethane thiosulfonate (MMTS) and 5,5-dithiobis(2-nitrobenzoate) (DTNB). This mutant enzyme was a starting point for probing by cysteine scanning the role of four residues of loop 3 in the catalysis and stability of the enzyme. Considering that the conservative substitution of either serine or alanine with cysteine would minimally alter the structure and properties of the environment of the residue in position 14, we made double mutants C14S/A69C, C14S/S71C, C14S/A73C, and C14S/S79C. Three of these double mutants were similar in their kinetic parameters to wild-type TbTIM and the single mutant C14S, but double mutant C14S/A73C showed a greatly reduced k cat. All enzymes had similar CD spectra, but all mutants had thermal stabilities lower than that of wild-type TbTIM. Intrinsic fluorescence was also similar for all enzymes, but the double mutants bound up to 50 times more 1-anilino-8-naphthalene sulfonate (ANS) and were susceptible to digestion with subtilisin. The double mutants were also susceptible to inactivation by sulfhydryl reagents. Double mutant C14S/S79C exhibited the highest sensitivity to MMTS and DTNB, bound a significant amount of ANS, and had the highest sensitivity to subtilisin. Thus, the residues at positions 73 and 79 are critical for the catalysis and stability of TbTIM, respectively.     

Highly specific inactivation of triosephosphate isomerase from Trypanosoma cruzi

We searched for molecules that selectively inactivate homodimeric triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the parasite that causes Chagas' disease. We found that some benzothiazoles inactivate the enzyme. The most potent were 3-(2-benzothiazolylthio)-propanesulfonic acid, 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid, and 2-(2-4(4-aminophenyl)benzothiazole-6-methylbenzothiazole-7-sulfonic acid. Half-maximal inactivation by these compounds was attained with 33, 56, and 8 microM, respectively; in human TIM, half-maximal inactivation required 422 microM, 3.3 mM, and 1.6 mM. In TcTIM, the effect of the benzothiazoles decreased as the concentration of the enzyme was increased. TcTIM has a cysteine (Cys 15) at the dimer interface, whereas human TIM has methionine in that position. In M15C human TIM, the benzothiazole concentrations that caused half-maximal inactivation were much lower than in the wild type. The overall findings suggest that the benzothiazoles perturb the interactions between the two subunits of TcTIM through a process in which the interface cysteine is central in their deleterious action.     

Characterization of Bacillus subtilis glutamine synthetase by limited proteolysis.

The inactivation of native glutamine synthetase (GS) from Bacillus subtilis by trypsin, chymotrypsin, or subtilisin followed pseudo-fast order kinetics. Trypsin cleaved the polypeptide chain of GS into two principal fragments, one of about 43,000 (Mr) and the other of smaller than 10,000. Chymotrypsin and subtilisin caused similar cleavage of GS. A large fragment (Mr 35,000) and one smaller than 10,000 were detected on SDS-PAGE. The nicked protein remained dodecameric, as observed on gel filtration, electrophoresis, and electron micrography. In the presence of glutamate, ATP, and Mn2+, the digestion of GS by each of the three proteases was retarded completely; however, the presence of one substrate, L-glutamate, ATP+Mn2+, or ATP+Mg2+ led to partial protection. The product, L-glutamine, did not retard but altered the susceptibility of the protease sensitive sites. Amino acid sequence analysis of the two smaller polypeptide fragments showed that the nicked region was around serine 375 and serine 311, respectively, and that both large fragments (43,000 and 35,000) were N-terminal polypeptides of GS. The serine 311 region was involved in the formation of the enzyme-substrate complex. Tyrosine 372 near serine 375 corresponded to tyrosine 397 which was adenylylated by adenyltransferase in Escherichia coli GS.     

Cold-labile hemolysin produced by limited proteolysis of theta-toxin from Clostridium perfringens

A nicked toxin whose hemolytic activity is temperature dependent was obtained by limited proteolysis of theta-toxin (Mr 54,000) with subtilisin. The nicked toxin (C theta) is a complex of two fragments: the N-terminal fragment (Mr 15,000) with basic isoelectric point and the C-terminal fragment (Mr 39,000) with the single cysteinyl residue of the toxin whose reduced form is essential for the hemolytic activity. C theta hemolyzes erythrocytes only at temperatures above 25 degrees C, whereas the native toxin hemolyzes them even at 10 degrees C. At temperatures below 25 degrees C, C theta does not hemolyze them although it does bind to membrane cholesterol and although no distinct difference was observed between the secondary structure of C theta and that of native toxin. It was found that C theta binds to the cells only in a reversible manner at low temperature, while the native one binds irreversibly to the cells within 10 min, which explains the cold lability of C theta on hemolysis. The structural basis of the cold lability was discussed through comparison of C theta with another nicked derivative of theta-toxin that was also obtained.     

Structural differences in triosephosphate isomerase from different species and discovery of a multitrypanosomatid inhibitor

We examined the interfaces of homodimeric triosephosphate isomerase (TIM) from eight different species. The crystal structures of the enzymes showed that a portion of the interface is markedly similar in TIMs from Trypanosoma cruzi (TcTIM), Trypanosoma brucei, and Leishmania mexicana and significantly different from that of TIMs from human, yeast, chicken, Plasmodium falciparum, and Entamoeba histolytica. Since this interfacial region is central in the stability of TcTIM, we hypothesized that it would be possible to find agents that selectively affect the stability of TIMs from the three trypanosomatids. We found that 6,6'-bisbenzothiazole-2,2' diamine in the low micromolar range causes a desirable irreversible inactivation of the enzymes from the three trypanosomatids and has no effect on the other five TIMs. Thus, the data indicate that it is possible to find compounds that induce selective inactivation of the enzymes from three different trypanosomatids.     

Perturbation of the dimer interface of triosephosphate isomerase and its effect on Trypanosoma cruzi

Background

Chagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM). The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM), and tested if they kill the parasite.

Methodology/Principal Findings

Dithiodianiline (DTDA) at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM). It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 Å resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture.

Conclusions/Significance

By targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.     

Location of intrinsic and inducible phenoloxidase activity in molluscan hemocyanin

The phenoloxidase (PO) activity of the hemocyanins (Hcs) from two molluscan species, the gastropod Helix pomatia (Hp) and the cephalopod Sepia officinalis (So), was studied. With catechol as substrate the Hcs showed a weak o-diPO activity, which was moderately enhanced on limited proteolysis with subtilisin. The sites in the Hc molecules mainly responsible for this activity were identified. The highest intrinsic o-diPO activity and also by far the highest level of induction were found in the functional units (FUs) Hp f and So g, isolated from Hp beta-Hc and So Hc (subunit 2), respectively. The results thus support the earlier conclusion, made on the basis of sequence homology between molluscan Hcs, that Hp f and So g are functional and structural analogues. The subtilisin treatment of Hp f also induced monoPO activity, considered to be at the origin of browning of the sample.     

Complex Formation of Microbial Alkaline Protease Inhibitor (S-SI) with Subtilisin BPN′ and Its Properties

Microbial alkaline protease inhibitor, S–SI, was investigated on the interaction with subtilisin BPN′ Inhibitory equivalent of S–SI to subtilisin BPN′ was determined that one molecule of S–SI (MW = 23,000) inhibited two molecules of subtilisin BPN′ (MW = 27,700). The S–SI-subtilisin BPN′ complex was isolated by gel filtration on Sephadex G–100 and rhombic crystals were obtained. DIP- and ZAGPCK-subtilisin BPN′ did not form such complex with S–SI. Homogeneity of the complex was determined by disc electrophoresis. The isoelectric point of the complex was pH 5.5. Assay of S–SI dissociated and amino acid analysis of the complex indicated that one subunit (a half molecule) of S–SI was combined with one molecule of subtilisin BPN′ From molecular weight determination, it was clarified that the complex was composed of one molecule (consist of 2 subunits) of S–SI and two molecules of subtilisin BPN′.     

Selective proteolysis of ovine lutropin or its beta subunit by endoproteinase Arg-C. Properties of the Arg beta 43 cleaved hormone

Ovine lutropin (oLH) and its beta subunit (oLH beta) were nicked by short-term incubations with endoproteinase Arg-C. Isolated oLH beta was rapidly nicked and converted from an Mr 18,000 band on sodium dodecyl sulfate-polyacrylamide gels to an Mr 13,000 band. Partial nicking of only the beta subunit in intact oLH was also observed as indicated by the appearance of small amounts of the Mr 13,000 band detected in Arg-C-treated oLH samples. The alpha subunit was protected by association with the beta subunit, but free alpha subunit was rapidly degraded. Sequence analysis of nicked oLH beta indicated that one of the peptide bonds on either side of Arg43 was cleaved by the protease, with a slight preference for the amino side of this residue. Nicked oLH beta was reassociated with oLH alpha, and the resulting dimer was separated from unrecombined subunits. The biologic activity of nicked oLH beta + oLH alpha in an LH radioligand assay was only 2% that of intact oLH.     

Internal motion of a tryptophan residue in Streptomyces subtilisin inhibitor: deuterium nuclear magnetic resonance in solution

Deuterium NMR spectroscopy was used to study internal motions of a deuterium-labeled single tryptophan (Trp) residue (per subunit) of Streptomyces subtilisin inhibitor (SSI) in solution. The free inhibitor with the five ring protons of the Trp replaced with deuterons showed a narrow resonance component (56 Hz) of about one-quarter of the total intensity, in addition to the broad resonance component (about 600 Hz) at 25 degrees C, showing that it exits in an equilibrium mixture of two conformers, in one of which the tryptophan side chain is highly mobile. In analogy to the two structures of SSI found in the crystal, these two conformers were attributed to the one in which the contact between the alpha-lobe and the beta-lobe of the subunit is tight and the other in which the same contact is loose. When SSI forms a complex with subtilisin BPN', the broad component becomes invisibly broad, but the narrow component increases with even further narrowing, suggesting that the binding to the enzyme favors the "loose" conformer over the "tight" conformer.     

The stability and formation of native proteins from unfolded monomers is increased through interactions with unrelated proteins

The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.