Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A.

Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.     

Microinjected ribonuclease A as a probe for lysosomal pathways of intracellular protein degradation

There are multiple pathways of intracellular protein degradation, and molecular determinants within proteins appear to target them for particular pathways of breakdown. We use red cell-mediated microinjection to introduce radiolabeled proteins into cultured human fibroblasts in order to follow their catabolism. A well-characterized protein, bovine pancreatic ribonuclease A (RNase A), is localized initially in the cytosol of cells after microinjection, but it is subsequently taken up and degraded by lysosomes. This lysosomal pathway of proteolysis is subject to regulation in that RNase A is taken up and degraded by lysosomes at twice the rate when serum is omitted from the culture medium. Subtilisin cleaves RNase A between residues 20 and 21, and the separated fragments are termed RNase S-peptide (residues 1–20) and RNase S-protein (residues 21–124). Microinjected RNase S-protein is degraded in a serum-independent manner, while RNase S-peptide microinjected alone shows a twofold increase in degradation in response to serum withdrawal. Furthermore, covalent linkage of S-peptide to other proteins prior to microinjection causes degradation of the conjugate to become serum responsive. These results show that recognition of RNase A and certain other proteins for enhanced lysosomal degradation during serum withdrawal is based on some feature of the amino-terminal 20 amino acids. The entire S-peptide is not required for enhanced lysosomal degradation during serum withdrawal because degradation of certain fragments is also responsive to serum. We have identified the essential region to be within residues 7–11 of RNase S-peptide (Lys-Phe-Glu-Arg-Gln; KFERQ). To determine whether related peptides exist in cellular proteins, we raised antibodies to the pentapeptide. Affinity-purified antibodies to KFERQ specifically precipitate 25–35% of cellular proteins, and these proteins are preferentially degraded in response to serum withdrawal. Computer analyses of known protein sequences indicate that proteins degraded by lysosomes at an enhanced rate in response to serum withdrawal contain peptide regions related, but not identical, to KFERQ. We suggest two possible peptide motifs related to KFERQ and speculate about possible mechanisms of selective delivery of proteins to lysosomes based on such peptide regions.     

Two distinct regions on the surface of an RNA-binding domain are crucial for RNase E function

Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67 and Lys-112 [corrected], is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The side-chains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67 and Lys-112 [corrected], which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity.     

Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor

The dissociation rate constant of the angiogenin-placental ribonuclease inhibitor complex was determined by measuring the release of free angiogenin from the complex in the presence of scavenger for free placental ribonuclease inhibitor (PRI). In 0.1 M NaCl, pH 6, 25 degrees C, this value is 1.3 X 10(-7) s-1 (t1/2 congruent to 60 days). The Ki value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is 7.1 X 10(-16) M. The corresponding values for the interaction of RNase A with PRI, determined by similar means, are both considerably higher: the dissociation rate constant is 1.5 X 10(-5) s-1 (t1/2 = 13 h), and the Ki value is 4.4 X 10(-14) M. Thus, PRI binds about 60 times more tightly to angiogenin than to RNase A. The effect of increasing sodium chloride concentration on the binding of PRI to RNase A was explored by Henderson plots. The Ki value increases to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl, suggesting the importance of ionic interactions. The mode of inhibition of RNase A by PRI was determined by examining the effect of a competitive inhibitor of RNase A, cytidine 2'-phosphate, on the association rate of PRI with RNase A. Increasing concentrations of cytidine 2'-phosphate decrease the association rate in a manner consistent with a competitive mode of inhibition.     

Peptidyl linkers for protein heterodimerization catalyzed by microbial transglutaminase

Specific peptidyl linkers that result in the heterodimerization of functional proteins, which is catalyzed by microbial transglutaminase from Streptomyces mobaraensis (MTG), were generated based on a ribonuclease S-peptide using site-directed mutagenesis. The peptidyl linkers designated as Lys-tag and Gln-tag were designed to possess sole reactive Lys or Gln residue that was amenable for selective Lys-Gln cross-linkage of different proteins. Green fluorescent protein variants, ECFP and EYFP, were employed as model proteins, and those Lys- and Gln-tags were fused to the N-termini of ECFP and EYFP, respectively. As a result, we succeeded in solely obtaining the ECFP-EYFP heterodimer without forming multiply cross-linked byproducts. It was found that the reactivity of peptidyl linkers varied according to the type of amino acid to be replaced. Peptidyl linkers with a basic amino acid (Arg) exhibited the highest reactivity in the cross-linking reaction, suggesting the cationic residue substrate preference of MTG. Kinetic analysis utilizing fluorescent resonance energy transfer (FRET), that is only observed upon the heterodimeric ECFP-EYFP conjugation, revealed that the amino acid replacement contributed to the acceleration of cross-linking reactions by increasing catalytic turnover (k(cat)), rather than substrate binding affinity (K(m)). Finally, using a ribonuclease S-protein, the manipulation of enzymatic protein cross-linking based on specific S-peptide:S-protein interactions was explored. Since newly designed Lys- and Gln-tags retained binding affinities to the S-protein, the heterodimerization was perfectly restrained by wrapping them with the S-protein. The results suggest the possibility of limited protein conjugation by tuning steric hindrance against the MTG. Tailoring enzymatic posttranslational modifications with either engineering peptidyl substrates or by taking specific peptide-protein interactions into consideration may facilitate the development of a new sequential protein conjugation method for the preparation of multifunctional protein.     

Kinetic characterization of two active mutants of placental ribonuclease inhibitor that lack internal repeats

Human placental ribonuclease inhibitor (PRI), a 50-kDa tight-binding inhibitor of angiogenin and pancreatic ribonuclease, consists predominantly of 7 internal repeats, each 57 residues long. Repeats 3 plus 4 (residues 144-257) or repeat 6 (residues 315-371) can be deleted to give mutant proteins, PRI delta 3-4 and PRI delta 6, respectively, that retain inhibitory activity [Lee, F. S., & Vallee, B. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883]. We describe here the isolation and characterization of these two active mutant proteins. Both inhibit the enzymatic activities of either angiogenin or bovine pancreatic ribonuclease A (RNase A) with a 1:1 stoichiometry, and the mode of inhibition of RNase A by either is competitive. PRI delta 3-4 binds to angiogenin and RNase A with Ki values of 0.72 and 170 pM, respectively The corresponding values for PRI delta 6 are 22 and 43 pM, respectively. Since recombinant PRI to angiogenin and RNase A with Ki values of 0.29 and 68 fM, respectively, deletion of repeats 3 plus 4 weakens both interactions 2500-fold while deletion of repeat 6 weakens them 76,000- and 630-fold, respectively. Therefore, either the deletion of these repeats has altered the conformation of the angiogenin/RNase binding site in PRI or the deleted repeats contribute directly to the binding site, or both. In addition, the tighter binding to angiogenin versus RNase A seen with native PRI has been preserved in PRI delta 3-4 but has been almost completely abolished in PRI delta 6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7-11 as the essential pentapeptide

We have identified a pentapeptide region of microinjected ribonuclease A that is required for enhanced degradation of this protein during serum withdrawal. We introduced reductively methylated [3H]ribonuclease A, [3H]ribonuclease S-protein (residues 21-124), and [3H]ribonuclease S-peptide (residues 1-20) into the cytosol of human fibroblasts by red cell-mediated microinjection and osmotic lysis of pinosomes. The degradative rates of ribonuclease A and ribonuclease S-peptide are increased 2-fold upon withdrawal of serum, while catabolism of ribonuclease S-protein is not regulated in this manner. Certain fragments of ribonuclease S-peptide are also degraded in a serum-dependent fashion (residues 1-14 and 4-13), while other fragments are not (residues 1-10 and 2-8). [3H]Ribonuclease S-peptide is cleaved into two smaller radioactive peptides during loading into red cell ghosts. We tentatively identified the larger fragment as residues 7-11 based on its molecular weight determined by Sephadex chromatography in the presence of 8 M urea combined with sequential Edman degradation to identify the position of radioactive lysines. The smaller peptide fragment appears to be the amino-terminal dipeptide, Lys-Glu, and/or residues 7-8, Lys-Phe. After microinjection into fibroblasts, the pentapeptide is degraded at an enhanced rate in the absence of serum, while degradation of the dipeptide is not affected. We confirmed that residues 7-11 constitute the larger hydrolysis product of S-peptide by synthesizing this pentapeptide and radiolabeling it by reductive methylation. It migrated at the expected position after Sephadex chromatography in 8 M urea and was further hydrolyzed only slightly during loading into red cells. Finally, degradation of this pentapeptide after injection into fibroblasts was enhanced 2-fold upon serum withdrawal. These results, combined with our other recent studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that the pentapeptide, Lys-Phe-Glu-Arg-Gln, targets microinjected ribonuclease A to lysosomes for enhanced degradation during serum deprivation.     

Purification and characterization of a new ribonuclease from fruiting bodies of the oyster mushroom Pleurotus ostreatus.

A ribonuclease (RNase), possessing an N-terminal sequence disparate from those of ribonucleases from other mushrooms and previously isolated Pleuotus ostreatus RNases, was purified from the fruiting bodies of the edible mushroom Pleurotus ostreatus. The N-terminal sequence of Pleurotus ostreatus RNase did not manifest homology even to a previously reported RNase from the same mushroom. The ribonuclease was adsorbed on CM-Sepharose and Mono S. It exhibited a molecular mass of 12 kDa in both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. The ribonuclease displayed an activity of 11490 U/mg on yeast tRNA. The highest ribonuclease activity was exhibited toward poly U, followed by poly A and poly C. No activity was shown toward poly G. The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C. It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 240 nM.     

A 'cassette' RNase: site-selective cleavage of RNA by RNase S equipped with RNA-recognition segment

RNase S is a unique protein comprising the non-covalent association of two components, the S-peptide and the S-protein. An RNA-recognition segment derived from the human immunodeficiency virus (HIV)-1 Rev protein was conjugated with the S-peptide to form a complex with the S-protein. The resulting RNase S bearing the RNA-recognition segment preferentially hydrolyzed a single position of the RNA stem-loop derived from the specific binding site for the Rev protein.     

Enzymatically active angiogenin/ribonuclease A hybrids formed by peptide interchange

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins.

The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate. One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values. Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution. These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein. This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease. The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease. This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group.     

N端缺失突变对核糖核酸酶抑制因子活性的影响

人胎盘核糖核酸酶抑制因子(HRI)是一种存在于细胞浆中的50 kD的酸性蛋白质,富含亮氨酸和半肤氨酸.作为胞浆蛋白可保护细胞不受外来胰RN白s。侵袭.HRI主要结构是由7个富含亮氨酸的重复序列组成,7个亮氨酸重复单位有规律环状排列使N端、c端在空间上较为接近.用PcR方法在HRI cl〕NAS,端去除30个核普酸,并将此缺失突变的HRI的cl〕NA片段构建于质粒pPIcgK,电击转化入毕赤酵母(Pi峨i。 Pasto汀S)Gslls中,进行分泌型表达.对表达产物进行亲和层析纯化.实验结果表明,N端缺失突变的HRI与RN白s。A的亲合力较野生型HRI降低1倍,但依然具有竞争性抑制RNas。A的活性,表明HRIN端10个氨基酸残基缺失后并未丧失其抑制活性.     

Tandemization endows bovine pancreatic ribonuclease with cytotoxic activity

Due to their ability to degrade RNA, selected members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol of target cells, where they degrade cellular RNA and cause cell death. The cytotoxic activity of most RNases, however, is abolished by the cytosolic ribonuclease inhibitor (RI). Consequently, the development of RNase derivatives with the ability to evade RI binding is a desirable goal. In this study, tandem enzymes consisting of two RNase A units that are bound covalently via a peptide linker were generated by gene duplication. As deduced from the crystal structure of the RNase A.RI complex, one RNase A unit of the tandem enzyme can still be bound by RI. The other unit, however, should remain unbound because of steric hindrance. This free RNase A unit is expected to maintain its activity and to act as a cytotoxic agent. The study of the influence of the linker sequence on the conformation and stability of these constructs revealed that tandemization has only minor effects on the activity and stability of the constructs in comparison to monomeric RNase A. Relative activity was decreased by 10-50% and the melting temperature was decreased by less than 2.5 K. Furthermore, the cytotoxic potency of the RNase A tandem enzymes was investigated. Despite an in vitro inhibition by RI, tandemization was found to endow RNase A with remarkable cytotoxic activity. While monomeric RNase A is not cytotoxic, IC(50) values of the RNase A tandem variants decreased to 70.3-12.9 microM. These findings might establish the development of a new class of chemotherapeutic agents based on pancreatic ribonucleases.     

Decavanadate inhibits catalysis by ribonuclease A

Pentavalent organo-vanadates have been used extensively to mimic the transition state of phosphoryl group transfer reactions. Here, decavanadate (V(10)O(28)6-) is shown to be an inhibitor of catalysis by bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry shows that the Kd for the RNase A decavanadate complex is 1.4 microM. This value is consistent with kinetic measurements of the inhibition of enzymatic catalysis. The interaction between RNase A and decavanadate has a coulombic component, as the affinity for decavanadate is diminished by NaCl and binding is weaker to variant enzymes in which one (K41A RNase A) or three (K7A/R10A/K66A RNase A) of the cationic residues near the active site have been replaced with alanine. Decavanadate is thus the first oxometalate to be identified as an inhibitor of catalysis by a ribonuclease. Surprisingly, decavanadate binds to RNase A with an affinity similar to that of the pentavalent organo-vanadate, uridine 2',3'-cyclic vanadate.     

Acetyl coenzyme A binding by chloramphenicol acetyltransferase: long-range electrostatic determinants of coenzyme A recognition.

The possible involvement of arginyl and lysyl side chains of chloramphenicol acetyltransferase (CAT) in binding coenzyme A (CoA) was studied by means of chemical modification, site-directed mutagenesis, variation in ionic strength, use of competitive inhibitors or substrate analogues, and X-ray crystallography. Unlike a number of enzymes, including citrate synthase, CAT does not employ specific ion pairs with the phosphoanionic centers of CoA to bind the acetyl donor, and arginyl residues play no role in recognition of the coenzyme. Although phenylglyoxal inactivates CAT reversibly, it does so by the formation of an unstable adduct with a thiol group, that of Cys-31 in the chloramphenicol binding site. The inhibitory effect of increasing ionic strength on kcat/Km(acetyl-CoA) can be explained by long-range electrostatic interactions between CoA and the epsilon-amino groups of Lys-54 and Lys-177, both of which are solvent-accessible. The epsilon-amino group of Lys-54 contributes 1.3 kcal.mol-1 to the binding of acetyl-CoA via interactions with both the 3'- and 5'-phosphoanions of CoA. Lys-177 contributes only 0.4 kcal.mol-1 to the productive binding of acetyl-CoA, mediated by long-range (approximately 14 A) interactions with the 5'-alpha- and -beta-phosphoanions of CoA. The combined energetic contribution of Lys-54 and Lys-177 to acetyl-CoA binding (1.7 kcal.mol-1) is less than that previously demonstrated (2.4 kcal.mol-1) for a simple hydrophobic interaction between Tyr-178 and the adenine ring of CoA (Day & Shaw, 1992).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of ribonuclease T2 from Aspergillus oryzae.

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mol-1 at 25 degrees C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 degrees C and 119.1 kcal.mol-1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNAse T2 in the native state is predominantly due to the slow rate of unfolding.     

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

High affinity RNase S-peptide variants obtained by phage display have a novel "hot-spot" of binding energy.

Using phage display mutagenesis, high affinity variants of RNase S-peptide were produced that bind to RNase S-protein over 100-fold more tightly than the wild type S-peptide. The S-peptide: S-protein interface was further characterized using "biased" phage display libraries, where each targeted residue was constrained to be either polar or nonpolar. The use of these tailored libraries placed constraints on the type of interactions present during affinity maturation process and allowed more amino acids to be randomized simultaneously. These results, in conjunction with kinetic association and dissociation constants determined by surface plasmon resonance (SPR), highlight the role of a single mutation (A5W) in increasing S-peptide binding affinity. High affinity S-peptide variants were only identified when tryptophan was present in the phage display library at position 5, suggesting that this residue is a "hot-spot" of binding energy in the high affinity variants. Analysis of SPR data in the presence of denaturant suggests that the increased affinity is a result of increased hydrophobic interactions in the transition state rather than a stabilization of helical structure.