Energetics of folding subtilisin BPN'.

Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. Here we document for the first time the in vitro folding of the mature form of subtilisin. Subtilisin was modified by site-directed mutagenesis to be proteolytically inactive, allowing the impediments to folding to be systematically examined. First, the thermodynamics and kinetics of calcium binding to the high-affinity calcium A-site have been measured by microcalorimetry and fluorescence spectroscopy. Binding is an enthalpically driven process with an association constant (Ka) equal to 7 x 10(6) M-1. Furthermore, the kinetic barrier to calcium removal from the A-site (23 kcal/mol) is substantially larger than the standard free energy of binding (9.3 kcal/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow (t1/2 = 1.3 h at 25 degrees C). Second, to measure the kinetics of subtilisin folding independent of calcium binding, the high-affinity calcium binding site was deleted from the protein. At low ionic strength (I = 0.01) refolding of this mutant requires several days. The folding rate is accelerated almost 100-fold by a 10-fold increase in ionic strength, indicating that part of the free energy of activation may be electrostatic. At relatively high ionic strength (I = 0.5) refolding of the mutant subtilisin is complete in less than 1 h at 25 degrees C. We suggest that part of the electrostatic contribution to the activation free energy for folding subtilisin is related to the highly charged region of the protein comprising the weak ion binding site (site B).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid folding of calcium-free subtilisin by a stabilized pro-domain mutant.

In vitro folding of mature subtilisin is extremely slow. The isolated pro-domain greatly accelerates in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In our studies of subtilisin, we are trying to answer two basic questions: why does subtilisin fold slowly without the pro-domain and what does the pro-domain do to accelerate the folding rate? To address these general questions, we are trying to characterize all the rate constants governing individual steps in the bimolecular folding reaction of pro-domain with subtilisin. Here, we report the results of a series of in vitro folding experiments using an engineered pro-domain mutant which is independently stable (proR9) and two calcium-free subtilisin mutants. The bimolecular folding reaction of subtilisin and proR9 occurs in two steps: an initial binding of proR9 to unfolded subtilisin, followed by isomerization of the initial complex into the native complex. The central findings are as follows. First, the independently stable proR9 folds subtilisin much faster than the predominantly unfolded wild-type pro-domain. Second, at micromolar concentrations of proR9, the subtilisin folding reaction becomes limited by the rate at which prolines in the unfolded state can isomerize to their native conformation. The simpliest mechanism which closely describes the data includes two denatured forms of subtilisin, which form the initial complex with proR9 at the same rate but which isomerize to the fully folded complex at much different rates. In this model, 77% of the subtilisin isomerizes to the native form slowly and the remaining 23% isomerizes more rapidly (1.5 s-1). The slow-folding population may be unfolded subtilisin with the trans form of proline 168, which must isomerize to the cis form during refolding. Third, in the absence of proline isomerization, the rate of subtilisin folding is rapid and at [proR9] 3 s-1. The implications of these results concerning why subtilisin folds slowly without the pro-domain are discussed.     

Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of protein folding

The high structural resolution of the main transition states for the formation of native structure for the six small proteins of which Phi-values for a large set of mutants have become available, barstar, barnase, chymotrypsin inhibitor 2, Arc repressor, the src SH3 domain, and a tetrameric p53 domain reveals that for the first 5 of these proteins: (1) Residues that belong to regular secondary structure have a significantly larger average fraction of native structural consolidation than residues in loops; (2) on the other hand, secondary and tertiary structures have built up to the same degree, or at least a high degree, but nonuniformly distributed over the molecule; (3) the most consolidated parts of each protein molecule in the transition state cluster together, and these clusters contain a significantly higher percentage of residues that belong to regular secondary structure than the rest of the molecule. These observations further reconcile the framework model with the nucleation-condensation mechanism for folding: The amazing speed of protein folding can be understood as caused by the catalytic effect of the formation of clusters of residues which have particularly high preferences for the early formation of regular secondary structure in the presence of significant amounts of tertiary structure interactions.     

Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.     

Mechanism of acid-induced folding of proteins

We have previously shown [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577] that beta-lactamase, cytochrome c, and apomyoglobin are maximally unfolded at pH 2 under conditions of low ionic strength, but a further decrease in pH, by increasing the concentration of HCl, refolds the proteins to the A state with properties similar to those of a molten globule state. To understand the mechanism of acid-induced refolding of protein structure, we studied the effects of various strong acids and their neutral salts on the acid-unfolded states of ferricytochrome c and apomyoglobin. The conformational transition of cytochrome c was monitored at 20 degrees C by using changes in the far-UV CD and in the Soret absorption at 394 nm, and that of apomyoglobin was monitored by changes in the far-UV CD. Various strong acids (i.e., sulfuric acid, perchloric acid, nitric acid, trichloroacetic acid, and trifluoroacetic acid) refolded the acid-unfolded cytochrome c and apomyoglobin to the A states as was the case with HCl. For both proteins neutral salts of these acids caused similar conformational transitions, confirming that the anions are responsible for bringing about the transition. The order of effectiveness of anions was shown to be ferricyanide greater than ferrocyanide greater than sulfate greater than thiocyanate greater than perchlorate greater than iodide greater than nitrate greater than trifluoroacetate greater than bromide greater than chloride.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of a diffusional protein folding reaction

The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB.     

Mechanism of the dihydroorotase reaction

Dihydroorotase (DHO) is a zinc metalloenzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate and dihydroorotate. A chemical mechanism was proposed on the basis of an analysis of the effects of pH, metal substitution, solvent isotope effects, mutant proteins, and alternative substrates on the enzyme-catalyzed reaction. The pH-rate profiles for the hydrolysis of dihydroorotate or thiodihydroorotate demonstrated that a single group from the enzyme must be unprotonated for maximal catalytic activity. Conversely, the pH-rate profiles for the condensation of carbamoyl aspartate to dihydroorotate showed that a single group from the enzyme must be protonated for maximal catalytic activity. The native zinc ions within the active site of DHO were substituted with cobalt or cadmium by reconstitution of the apoenzyme with divalent cations in the presence of bicarbonate. The ionizations observed in the pH-rate profiles were dependent on the specific metal ion bound to the active site. Mutation of the residue (Asp-250) that hydrogen bonds to the bridging hydroxide (or water) resulted in the loss of catalytic activity. These results are consistent with the formation of a hydroxide bridge between the two divalent cations that functions as the nucleophile during the hydrolysis of dihydroorotate. In addition, Asp-250 is postulated to shuttle the proton from the bridging hydroxide to the leaving group amide during hydrolysis of dihydroorotate. The X-ray crystal structure of DHO showed that the exocyclic alpha-carboxylate of dihydroorotate is bound to the protein via electrostatic interactions with Arg-20, Asn-44, and His-254. Mutation of these residues resulted in the loss of catalytic activity, indicating that these residues are critical for substrate recognition. The thio analogue of dihydroorotate was found to be a good substrate of the enzyme. A comprehensive chemical mechanism for DHO was proposed on the basis of the experimental findings in this study and the X-ray crystal structure.     

Stoichiometric complexation of streptomyces subtilisin inhibitor and subtilisin

Subtilisin (Sbt) andStreptomyces subtilisin inhibitor (SSI) were analyzed either alone or together using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). With all ratios of Sbt to SSI tested, the proteins formed a stoichiometric complex, and migrated abnormally at the top of the gel. Electroblotting and amino acid sequence analysis of the complex band showed both Sbt and SSI present at approximately equal molar ratios. When excess Sbt was present, it migrated as a free but still folded form slightly above the band corresponding to the complex. When excess SSI was present, it migrated as several species with molecular weights smaller than the intact form; in fact, the sequences of some of these species indicated that they lacked different amounts of N-terminal and possibly C-terminal residues.     

Mechanism of induced folding: Both folding before binding and binding before folding can be realized in staphylococcal nuclease mutants

A considerable number of functional proteins are unstructured under physiological condition. These "intrinsically disordered" proteins exhibit induced folding when they bind their targets. The induced folding comprises two elementary processes: folding and binding. Two mechanisms are possible for the induced folding: either folding before binding or binding before folding. We found that these two mechanisms can be distinguished by the target-concentration dependence of folding kinetics. We also created two types of mutants of staphylococcal nuclease showing the different inhibitor-concentration dependence of induced folding kinetics. One mutant obeys the scheme of binding before folding, while the other the folding before binding. This is the first experimental evidence demonstrating that both mechanisms are realized for a single protein. Binding before folding is possible, when the protein lacks essential nonlocal interaction to stabilize the native conformation. The results cast light on the protein folding mechanism involved in the intrinsically disordered proteins.     

Mechanism of the glutamate dehydrogenase reaction

The data concerning the chemical and kinetic mechanisms of the glutamate dehydrogenase reaction have been reviewed. Based on the differences between two catalytically active glutamate dehydrogenase conformations induced by the substrates as well as on some other evidence, it has been proposed that the amino groups of lysine residues 27 and 126 in the beef liver enzyme are interchangeable depending on the direction of the glutamate dehydrogenase reaction.     

Mechanism of the microtubule GTPase reaction

The rate of GTP hydrolysis by microtubules has been measured at tubulin subunit concentrations where microtubules undergo net disassembly. This was made possible by using microtubules stabilized against disassembly by reaction with ethylene glycol bis-(succinimidylsuccinate) (EGS) as sites for the addition of tubulin-GTP subunits. The tubulin subunit concentration was varied from 25 to 90% of the steady state concentration, and there was no net elongation of stabilized microtubule seeds. The GTPase rate with EGS microtubules was linearly proportional to the tubulin-GTP subunit concentration when this concentration was varied by dilution and by using GDP to compete with GTP for the tubulin E-site. The linear dependence of the rate is consistent with a GTP mechanism in which hydrolysis is coupled to the tubulin-GTP subunit addition to microtubule ends. It is inconsistent with reaction schemes in which: microtubules are capped by a single tubulin-GTP subunit, which hydrolyzes GTP when a tubulin-GTP subunit adds to the end; hydrolysis occurs primarily in subunits at the interface of a tubulin-GTP cap and the tubulin-GDP microtubule core; hydrolysis is not coupled to subunit addition and occurs randomly in subunits in a tubulin-GTP cap. It was also found that GDP inhibition of the microtubule GTPase rate results from GDP competition for GTP at the tubulin subunit E-site. There is no additional effect of GDP on the GTPase rate resulting from exchange into tubulin subunits at microtubule ends.     

Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding

The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3 A resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central alphabetaalpha substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca2+ binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca2+ for folding of the mature domain and completion of the folding process prior to autoprocessing.     

Mechanism of the thrombin-platelet reaction]

In the course of investigations concerning the mechanism of the thrombin platelet interaction the conditions for the appearance of a refractory behaviour of platelets towards thrombin were examined. As investigation of the platelet reactivity by determining the amine liberation showed that a continuous or discontinuous addition of slight amounts of thrombin will cause the refractory condition. The findings were analyzed in view of the interaction between the dissolved enzyme and the fixed substrate. It is assumed that the number of receptors diminished after the first thrombin contact will cause a decrease of the receptor occupation speed on the platelet surface, thus leading to a decreased platelet reactivity.