Thermodynamics of denaturation of complexes of barnase and binase with barstar

Differential scanning calorimetry was used to study the thermodynamics of denaturation of protein complexes for which the free energy stabilizing the complexes varied between -8 and -16 kcal/mol. The proteins studied were the ribonucleases barnase and binase, their inhibitor barstar and mutants thereof, and complexes between the two. The results are in good agreement with the model developed by Brandts and Lin for studying the thermodynamics of denaturation for tight complexes between two proteins which undergo two-state thermal unfolding transitions.     

The heat denaturation of bacterial ribonuclease studied by infrared spectroscopy]

The thermal denaturation of bacterial ribonuclease in the interval of pH 2.5-7.0 has been investigated by means of infra-red spectroscopy method. The protein melting for pH 2.5 begins at the temperature 25 degrees C and is accompanied by secondary protein structure reconstruction, partially destroying native beta-structure and leading to new denatured conformation appearance of different types of beta-turns. Spectral changes for pH 3.5 and 7.0 are significantly less in the same frequency areas. At the temperature more than 50 degrees C protein aggregation takes place with inter-molecule-beta-form formation.     

Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of beta-lactoglobulin

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.     

pH dependence of the stability of barstar to chemical and thermal denaturation.

Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F<->U mechanism and yields identical values for delta GU, the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 +/- 0.5 kcal.mol-1 at 25 degrees C. Thermal denaturation of barstar also follows a two-state F<->U unfolding transition at pH 7, and the value of delta GU at 25 degrees C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 +/- 0.2, is not observed in H17Q, a His 17-->Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 +/- 0.2, is not detected in BSCCAA, the Cys 40 Cys 82-->Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 +/- 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brownian dynamics simulation of the association of Bacillus amyloliquefaciens ribonuclease (barnase) and Bacillus intermedius ribonuclease (binase) with barstar

A comparative study of the association of two ribonucleases, barnase and binase, with the polypeptide inhibitor barstar has been performed by the Brownian dynamics simulation method. It was shown that the method adequately reproduced the dependence of the association rate on pH and ionic strength of solution and the influence of mutations of some ribonuclease amino acids. Two types of energetically favorable complexes of binase-barstar encounter were determined. In the type I complex, the amino acids of binase active center take part in the complex formation. In the second complex, the active center is free. It was supposed that the temporary binding of barstar into complex of type II is competitive relative to the inhibition reaction. This can partially explain the decrease in the rate of binase inhibition as compared with the corresponding reaction of barnase.     

The role of the single tryptophan residue in the structure and function of ribonuclease T1

The previously reported method for the preparation of Kyn 59-RNase T1 and NFK 59-RNase T1 has been improved, and these two proteins have been obtained in high purity. Kyn 59-RNase T1, fully active for the hydrolysis of GpA and GpC, emitted a 35-fold-enhanced fluorescence of kynurenine relative to acetylnurenine amide with an emission maximum at 455 nm upon excitation at 380 nm. The polarity of the environment of Kyn 59 estimated from the emission maximum corresponded to a dielectric constant of 6. Upon excitation at 325 nm, NFK 59-RNase T1, less active than Kyn 59-RNase T1, exhibited a quenched N'-formylkynurenine fluorescence with an emission maximum at 423 nm, from which the value of 12 was obtained as the dielectric constant of the surroundings of residue 59. In both modified proteins, distinct tyrosine fluorescence appeared on excitation at 280 nm. The detection of an energy transfer from tyrosine to residue 59 suggests that the tertiary structure is very similar in Kyn 59-RNase T1 and native RNase T1. With guanidine hydrochloride, Kyn 59-RNase T1 was less stable than the native protein. Carboxymethylation at Glu 58 was shown to stabilize the active site of the modified enzyme. Based on the information collected for Kyn 59-RNase T1, the local environment and possible roles of the sole tryptophan residue in RNase T1 are discussed.     

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.     

Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone.

The fluorescence-monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 degrees C, have been quantitatively analyzed using a 3-state mechanism: U(S)<-->UF<-->N. U(S) and UF are slow-refolding and fast-refolding unfolded forms of barstar, and N is the native protein. U(S) and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47-Pro 48 bond. The 3-state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3-state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio U(S):UF (K21) and the values of the rate constants of the U(S)-->UF and UF-->U(S) reactions, k12 and k21, respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 +/- 0.1, and k12 and k21 have values of 5.3 x 10(-3) s-1 and 11.2 x 10(-3) s-1, respectively. Double-jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K21, k12, and k21 obtained from the 3-state analysis and thereby the validity of the 3-state model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design of multivalent complexes using the barnase*barstar module

The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185(HER2-ECD) 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.     

Studies on the mechanism of inhibition of bacterial ribonuclease P by aminoglycoside derivatives

Ribonuclease P (RNase P) is a Mg²⁺-dependent endoribonuclease responsible for the 5′-maturation of transfer RNAs. It is a ribonucleoprotein complex containing an essential RNA and a varying number of protein subunits depending on the source: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Since bacterial RNase P is required for viability and differs in structure/subunit composition from its eukaryal counterpart, it is a potential antibacterial target. To elucidate the basis for our previous finding that the hexa-arginine derivative of neomycin B is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, we synthesized hexa-guanidinium and -lysyl conjugates of neomycin B and compared their inhibitory potential. Our studies indicate that side-chain length, flexibility and composition cumulatively account for the inhibitory potency of the aminoglycoside-arginine conjugates (AACs). We also demonstrate that AACs interfere with RNase P function by displacing Mg²⁺ ions. Moreover, our finding that an AAC can discriminate between a bacterial and archaeal (an experimental surrogate for eukaryal) RNase P holoenzyme lends promise to the design of aminoglycoside conjugates as selective inhibitors of bacterial RNase P, especially once the structural differences in RNase P from the three domains of life have been established.     

X-ray crystallographic studies of the denaturation of ribonuclease S.

In an attempt to view the onset of urea denaturation in ribonuclease we have collected X-ray diffraction data on ribonuclease S crystals soaked in 0, 1.5, 2, 3, and 5 molar urea. At concentrations above 2 M urea, crystals were stabilized by glutaraldehyde crosslinking. We have also collected data on ribonuclease S crystals at low pH in an attempt to study the onset of pH denaturation. The resolution of the datasets range from 1.9 to 3.0 A. Analysis of the structures reveals an increase in disorder with increasing urea concentration. In the 5 M urea structure, this increase in disorder is apparent all over the structure but is larger in loop and helical regions than in the beta strands. The low pH structure shows a very similar pattern of increased disorder. In addition there is a major change in the position of the main chain (> 1 A) in the 65-72 turn region. This region has previously been shown to be involved in one of the initial steps of unfolding in the reduction of ribonuclease A. Crystallographic analyses in the presence of denaturant, when combined with controlled crosslinking, can thus provide detailed structural information that is related to the initial steps of unfolding in solution. Proteins 1999;36:282-294.     

Structure-cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants

Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer. In the cytosolic reducing environment, the dimeric form in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor, a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor. On the basis of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity.     

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.     

The thermal denaturation of ribonuclease A in aqueous-methanol solvents.

Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.