Biomedical subjects

J Eder

Publications and source records attributed to J Eder.

At least 19 recordsLinked to original sources

PubMed2004

QTL mapping for European corn borer resistance ( Ostrinia nubilalis Hb.), agronomic and forage quality traits of testcross progenies in early-maturing European maize ( Zea mays L.) germplasm.

In hybrid breeding the performance of lines in hybrid combinations is more important than their performance per se. Little information is available on the correlation between individual line and testcross (TC) performances for the resistance to European corn borer (ECB, Ostrinia nubilalis Hb.) in maize ( Zea mays L.). Marker assisted selection (MAS) will be successful only if quantitative trait loci (QTL) found in F(2) derived lines for ECB resistance are still expressed in hybrid combinations. The objectives of our study were: (1) to identify and characterize QTL for ECB resistance as well as agronomic and forage quality traits in a population of testcrossed F(2:3) families; (2) to evaluate the consistency of QTL for per se and TC performances; and (3) to determine the association between per se and TC performances of F(2:3) lines for these traits. Two hundred and four F(2:3) lines were derived from the cross between maize lines D06 (resistant) and D408 (susceptible). These lines were crossed to D171 and the TC progenies were evaluated for ECB resistance and agronomic performance in two locations in 2000 and 2001. Using these TC progenies, six QTL for stalk damage rating (SDR) were found. These QTL explained 27.4% of the genotypic variance in a simultaneous fit. Three QTL for SDR were detected consistently for per se and TC performance. Phenotypic and genotypic correlations were low for per se and TC performance for SDR. Correlations between SDR and quality traits were not significant. Based on these results, we conclude that MAS will not be an efficient method for improving SDR. However, new molecular tools might provide the opportunity to use QTL data as a first step to identify genes involved in ECB resistance. Efficient MAS procedures might then be based on markers designed to trace and to combine specific genes and their alleles in elite maize breeding germplasm.

Alleles↗

PubMed2001

Intact T-cell regenerative capacity in childhood acute lymphoblastic leukemia after remission induction therapy.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a bone marrow disease. This may adversely affect the capacity of T cells to recover from chemotherapy-induced T-cell depletion and thus contribute to the prevailing immune deficiency in ALL patients. PROCEDURE: We tested the capacity of T-cells to regenerate in 18 ALL children in first clinical remission (median age 4.2 years) at the time of hematologic reconstitution after BFM-ALL induction therapy (treatment-free interval 22 days, median; range 12 to 52 days). All patients had experienced a period of leukopenia (white blood cell count [WBC] <0.95 x 10(9)/l, median) during the final four weeks of induction therapy. T-cells and T-cell subsets were examined by FACS. RESULTS: At the time of investigation the WBC was near normal (3.5 x 10(9)/l, median). Surprisingly, most cases (78%) showed a complete regeneration of T-cells and its subsets including 1) normal total (CD3+) T-cells (1635/microl, median; range 756-3440/microl); 2) normal T-helper (CD4+) cells (697/microl, median; range 128-1523/microl); and 3) normal T-cytotoxic/suppressor (CD8+) cells (686/microl, median; range 348-1540/microl). Eight patients achieved a normal CD4+/CD8+ ratio (0.8, median). Subset analyses of T-helper cells revealed a normal proportion of CD4+CD45RA+ cells (52%, median) in all but one patient below the age of 6 years, indicating an intact residual thymic activity. No correlation was observed between age at diagnosis and a normal CD4+ count (r = 0.086) or between a normal CD4+ count and a normal proportion of CD4+CD45RA+ cells r = 0.136). A long-term survey in four patients showed altered T-cells after reinduction and during maintenance therapy. CONCLUSIONS: The findings suggest that ALL per se does not inhibit T-cell regenerative capacity. Thus, the frequently observed longlasting impairment of the T-cell system in ALL is attributable to the treatment rather than to the underlying disease.

Adolescent↗

PubMed2000

Requirement of residual thymus to restore normal T-cell subsets after human allogeneic bone marrow transplantation.

BACKGROUND: To determine the effect of residual thymic activity in reconstituting the T-cell system after T cell-depleting therapy, we monitored T-cell subsets of a unique thymectomized cancer patient in comparison to thymus-bearing patients after allogeneic bone marrow transplantation (BMT). METHODS: T cells and T-cell subsets previously shown in murine studies to be regulated by the thymus were analyzed by FACS from 6 to >48 months after BMT. The investigation of thymus-bearing patients included 32 examinations of 9 children and 14 adults. None of the investigated cases had severe graft-versus-host disease or severe infections when examined. RESULTS: In the thymectomized host, T-cell regeneration occurred by donor cell expansion and was characterized by two prominent features: (i) a persistent failure to regenerate naive (CD45RA+) T-helper cells (14%, median), consistent with the recently developed concept of a thymus-dependency; and (ii) persistently elevated proportions of CD3+CD4-CD8- cells (double-negative cells, median 29%), which were identified in T cell receptor (TCR)gamma delta+ (22%, median of CD3+ cells, 88% double negatives) but also TCRalpha beta+ T-cell populations (78%, median of CD3+ cells, 17% double negatives). In thymus-bearing patients, 10 of 12 and 6 of 14 examinations of children and adults, respectively, performed later than 12 months after BMT showed the proportion of CD4+CD45RA+ cells appropriate for age (>52% and >28% in children and adults, respectively). Elevated double-negative cells (>10%) were found in only three patients, but none had elevated double-negative cells with a TCRalpha beta+ phenotype. CONCLUSION: Residual thymic activity might, in addition to its well-established role for regenerating naive T-helper (CD4+CD45RA+) cells, control the expansion of double-negative cells. A normal T-cell subset regeneration in a proportion of thymus-bearing adult hosts indicates the potential of an effective residual thymic activity even beyond childhood.

Adolescent↗

PubMed1999

An antigenic HIV-1 peptide sequence engineered into the surface structure of transferrin does not elicit an antibody response.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein such as human serum transferrin (HST). However, a potential drawback is that the HST may increase the immunoreactivity of the peptide, in the same way that carrier proteins can be used to generate highly immunogenic peptide hapten conjugates. In this study we have generated a recombinant HST carrier protein that contains a peptide substrate of HIV-1 protease (VSQNYPIVL). The protein retained native HST function, and the peptide was surface exposed since it was immunoreactive in native dot blots, and was cleaved by HIV-1 protease. Immunisation of rabbits with the recombinant protein elicited only a very poor anti-peptide immune response. In contrast, strong anti-peptide immune responses were raised against both the peptide alone, and a chemical conjugate of the peptide with HST. These data demonstrate that it is possible to attenuate the immune response normally directed against an immunogenic peptide sequence by engineering into a surface exposed loop of HST. These findings may have an important impact on the future design of peptide delivery systems.

Animals↗

PubMed1999

Transferrin trojan horses as a rational approach for the biological delivery of therapeutic peptide domains.

One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein, such as human serum transferrin. To examine whether this is feasible, a peptide sequence cleavable by the human immunodeficiency virus type 1 protease (VSQNYPIVL) was inserted into various regions of human serum transferrin, and the resultant proteins were tested for function. Experimentally, molecular modeling was used to identify five candidate insertion sites in surface exposed loops of human serum transferrin that were distant from biologically active domains. These insertions were cloned using polymerase chain reaction mutagenesis, and the proteins were expressed using a baculovirus expression vector system. Analysis of the mutant proteins provided a number of important findings: (a) they retained native human serum transferrin function, (b) the inserted peptide sequence was surface exposed, and most importantly, (c) two of these mutants could be cleaved by human immunodeficiency virus-1 protease. In conclusion, this investigation has validated the use of human serum transferrin as a carrier protein for functional peptide domains introduced into its structure using protein engineering. These findings will be useful for developing a novel class of therapeutic agents for a broad spectrum of diseases.

Anti-HIV Agents↗

PubMed1999

The kinetics of association and phosphorylation of IkappaB isoforms by IkappaB kinase 2 correlate with their cellular regulation in human endothelial cells.

Activation of the transcription factor NF-kappaB depends on the specific dual phosphorylation of its inhibitor protein IkappaB by the homologous cytokine-inducible IkappaB kinases 1 and 2 (IKK1/2). Various IkappaB isoforms exist: IkappaBalpha, IkappaBbeta1/2 (two alternative splice variants), and IkappaBepsilon. However, the individual relevance and the specific regulation of these isoforms is not well-understood. We have studied the direct interaction of recombinant IkappaBalpha, IkappaBbeta1, IkappaBbeta2, and IkappaBepsilon with the recombinant homodimeric IKK2. Fluorescence-based active site titration revealed that each IKK2 dimer contains two binding sites for IkappaB. By using surface plasmon resonance analysis, we found that all IkappaB proteins interact with the IKK2 dimer following a noncooperative binding mechanism. Further, the four IkappaB proteins bind to the kinase with equilibrium dissociation constants (KD) in the range of 50-300 nM; the association rate constants for all IkappaB isoforms with IKK2 were between 6.0 x 10(3) and 22.5 x 10(3) M-1 s-1, and the dissociation rate constants were between 1.25 x 10(-3) and 1.75 x 10(-3) s-1. This high-affinity binding suggests that the previously observed preassociation of all analyzed IkappaB proteins with the biochemically purified 700 kDa IkappaB kinase (IKK) complex is based on a direct enzyme-substrate association between the various IkappaB isoforms and the IKK proteins. The apparent catalytic efficiencies (kcat/KM) of IKK2 for IkappaBalpha, IkappaBbeta1, IkappaBbeta2, and IkappaBepsilon were 22 x 10(3), 10 x 10(3), 5.4 x 10(3), and 8.5 x 10(3) s-1 M-1, respectively, with KM values ranging between 1.7 x 10(-6) and 3.2 x 10(-6) M and kcat values ranging between 1.5 x 10(-2) and 3.7 x 10(-2) s-1. The relative affinities and catalytic efficiencies of IKK2 for the IkappaB isoforms were also reflected by the kinetics observed for the TNF-induced, phosphorylation-dependent degradation of the alpha, beta1, beta2, and epsilon isoforms of IkappaB in human umbilical vein endothelial cells. Therefore, differential regulation of the IkappaB isoforms in some cell types is not a direct result of the IKK activity, but appears to be due to parallel events.

DNA-Binding Proteins↗

PubMed1999

All three IkappaB isoforms and most Rel family members are stably associated with the IkappaB kinase 1/2 complex.

Nuclear factor kappa B (NF-kappaB) is an important transcription factor for the genes of many pro-inflammatory proteins and is strongly activated by the cytokines interleukin-1 and tumor necrosis factor (TNF)alpha under various pathological conditions. In nonstimulated cells, NF-kappaB is present in the cytosol where it is complexed to its inhibitor IkappaB. Activation of NF-kappaB depends on the signal-induced phosphorylation of IkappaB by specific IkappaB kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF-stimulated and okadaic-acid-stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in IkappaBalpha which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known IkappaB kinases: the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1), the IkappaB kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre-assembled kinase-substrate complex in which the IKKs are directly or indirectly associated with several NF-kappaB-related and IkappaB-related proteins: RelA, RelB, cRel, p100, p105, Ikappa Balpha, Ikappa Bbeta and Ikappa Bepsilon. The existence of stable kinase-substrate complexes, the presence of all three known IkappaB isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating Ikappa Balpha-, Ikappa Bbeta- and Ikappa Bepsilon-derived peptides at the respective degradation-relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF-kappaB species. In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C-terminal PEST sequence of Ikappa Balpha, we observed efficient phosphorylation of serine 32 in Ikappa Balpha by the purified endogenous CK II complex. Therefore, both p90rsk1 and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation.

Casein Kinases↗

PubMed1997

Tumour necrosis factor alpha and interleukin 1 signalling: do MAPKK kinases connect it all?

The potent pro-inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and interleukin 1 (IL-1) are capable of triggering biologically similar effects through activation of the same set of transcription factors. Based on recent findings it is now becoming evident that certain members of the mitogen-activated protein kinase kinase (MAPKK) kinase protein family serve to integrate the individual signal transduction pathways that are initiated by the two cytokines into an array of parallel and common signalling cascades. The link between the receptor proximal, signal-specific intracellular events and the common MAPKK kinases appears to be made by a new class of proteins known as TNF receptor associated factors (TRAFs). Here, Jörg Eder describes how TNF-alpha and IL-1 use different, pathway-specific TRAFs to activate the same MAPKK kinase-controlled cascades.

Animals↗

PubMed1996

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay.

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion binding capacity of the chelators.

Chelating Agents↗

PubMed1995

Isolation and structural characterization of different isoforms of the hypusine-containing protein eIF-5A from HeLa cells.

Posttranslational modification of a specific lysine residue in eukaryotic initiation factor 5A (eIF-5A) is essential for cell viability and proliferation. The product of this modification is hypusine, an amino acid unique to eIF-5A. We have purified and characterized one major and three minor isoforms of human eIF-5A from HeLa cells. The main form, which accounts for approximately 95% of the total eIF-5A, carries hypusine at position 50 and is amino-terminally acetylated as determined by amino acid composition analysis and electrospray ionization mass spectrometry. Analytical gel filtration indicates that this protein variant possesses a native apparent molecular weight that lies between that expected for a monomeric and dimeric form. Nevertheless, several experiments confirm this protein to be monomeric. It is further shown that eIF-5A have well-defined secondary structure. Both the far-UV circular dichroism spectrum as well as secondary structure predictions using different algorithms suggest this protein to have predominantly beta-sheet structure. Two plausible models for the packing of the secondary structure elements are presented. In contrast to the main form, all three minor isoforms of eIF-5A are characterized by acetylation of the epsilon-amino group of lysine at position 47. The minor isoforms are distinguishable by their state of modification of the lysine residue at position 50. Whereas the main form occurs in both the cytoplasmic and the nuclear fraction of HeLa cells, the minor isoforms were not detectable in the preparation of the nuclear fraction. Therefore, acetylation of lysine at position 47 might play a controlling role in the distribution of the minor isoforms to the nucleus.

Amino Acid Sequence↗

PubMed1995

The polypeptide chain of eukaryotic initiation factor 5A occurs in two distinct conformations in the absence of the hypusine modification.

Eukaryotic initiation factor 5A (eIF-5A) requires posttranslational modification of lysine at position 50 to hypusine for its biological activity. We have expressed an unmodified variant of eIF-5A in Escherichia coli and show that it has structural properties different from those of the native protein in terms of its near- and far-UV circular dichroism spectra and its equilibrium unfolding transition with guanidinium chloride. In contrast to the hypusine-modified protein, which unfolds in a two-state process, the complex unfolding transition of unmodified eIF-5A suggests that this variant occurs in two differently folded conformations, F1 and F2. Both conformations are populated under near-physiological conditions at a ration of 60 to 40, respectively. Equilibrium unfolding consists of parallel events: unfolding of F1 to one or several intermediate states (I), and unfolding of F2 to the unfolded state (U). Although the establishment of each of these individual equilibria is fast, the interconversion is slow at guanidinium chloride concentrations between 0 M and 3 M. Kinetic analysis reveals activation energies of 24.3 kcal mol-1 for the reaction of F1 and F2 and 24.1 kcal mol-1 for the reaction of F2 to F1. Both F1 and F2 possess well-defined secondary and tertiary structure. However, the tertiary structures of the two conformations differ as indicated by their distinct near-UV circular dichroism spectra. These differences may be restricted to the C-terminal part of the protein as 2-dimensional 1H-NMR spectra of unmodified eIF-5A reveal no doubled set of proton resonances for aromatic amino acid and histidine residues, of which almost all are located in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, Gel↗

PubMed1995

Purification and characterization of human deoxyhypusine synthase from HeLa cells.

Post-translational modification of a specific lysine residue in eukaryotic initiation factor 5A is essential for cell viability. The amino acid hypusine, which is the product of this modification, is derived in two subsequent enzyme-catalyzed reactions. We have purified and characterized the enzyme responsible for the first step in hypusine modification, deoxyhypusine synthase, from HeLa cells. The human enzyme is multimeric with a native apparent molecular weight of 150,000 consisting of subunits of 41,000. The amino acid sequences of its peptide fragments share high sequence identity with a hypothetical protein (YHRO68w) on chromosome VIII of Saccharomyces cerevisiae. This protein appears to be the deoxyhypusine synthase of yeast.

Amino Acid Sequence↗

PubMed1995

Pro-sequence-assisted protein folding.

Many proteins, including proteases and growth factors, are synthesized as precursors in the form of pre-pro-proteins. Whereas the pre-sequences usually act as signal peptides for transport, the pro-sequences of an increasing number of these proteins have been found to be essential for the correct folding of their associated proteins. In contrast to the action of molecular chaperones, pro-sequences appear to catalyse the protein-folding reaction directly. The similarity between the pro-sequence-assisted folding mechanisms of different proteases supports the hypothesis that a common folding mechanism has developed through convergent evolution. Further, the frequent requirement of the pro-sequences for both folding and intracellular transport or secretion suggests that these two functionalities are intimately related.

Biological Evolution↗

PubMed1994

Variants of subtilisin BPN' with altered specificity profiles.

A strategy for increasing the size of the S4 binding pocket was used to improve the specificity of subtilisin BPN' toward substrates with large hydrophobic P4 side chains. This approach involves single and double amino acid replacements at positions 104, 107, and 126. Previously, alteration of I107 to glycine has been found to increase the specificity of subtilisin toward leucine, isoleucine, and phenylalanine as P4 residues by up to 214-fold. Replacement of Y104 by alanine also yields a similar improvement in specificity. However, this subtilisin variant favors isoleucine and phenylalanine over leucine. When L126 was replaced by valine, alanine, and glycine, respectively, only the L126A subtilisin variant, which possesses a 28-fold-increased catalytic efficiency for isoleucine compared with all other substrates tested, showed a significantly improved specificity profile. As inferred from the double-mutant enzymes I107G/L126V, I107G/L126A, and I107G/Y104A, none of the effects of the single amino acid replacements on the kinetic parameters are additive. The I107G/L126V mutant subtilisin has the largest improvement in P4 substrate specificity reported so far: kcat/KM is increased 340-fold for leucine compared to alanine. By contrast, the specificity profile of the I107G/Y104A mutant enzyme is impaired in comparison with that of the corresponding single mutants. Therefore, the design of high-specificity subtilisin variants through the combination of single amino acid replacements in the S4 pocket appears to be nontrivial due to the interference of the introduced structural changes.

Amino Acid Sequence↗

PubMed1993

Hydrolysis of small peptide substrates parallels binding of chymotrypsin inhibitor 2 for mutants of subtilisin BPN'.

Variants of subtilisin BPN' that possess improved specificity towards isoleucine compared with alanine at the P4 position of small peptide substrates, were analysed for their ability to bind chymotrypsin inhibitor 2. The binding of the inhibitor with isoleucine (wild-type) and with alanine as the P4 residue parallels the hydrolysis of tetrapeptide substrates. There is a linear relationship between the free energy of binding of the transition state of the substrate and the free energy of binding of the inhibitor with a slope of 2.0. The data suggest that the inhibitor uses predominantly ground state rather than transition state binding energy.

Amino Acid Sequence↗

PubMed1993

Folding of subtilisin BPN': role of the pro-sequence.

Subtilisin BPN' is an extracellular serine protease from Bacillus amyloliquefaciens that requires an N-terminal 77 amino acid pro-sequence for correct folding of the catalytic domain. We have expressed an inactive, stable pro-subtilisin variant in Escherichia coli and show that it has structural properties similar to native subtilisin in terms of its near- and far-UV circular dichroism spectra, its compactness, and its capacity to bind calcium ions stoichiometrically. Unlike subtilisin, the pro-subtilisin variant unfolds reversibly with guanidinium chloride, and unfolding occurs via a folding intermediate. This intermediate is similar to the metastable intermediate state recently found for folding of subtilisin in the absence of the pro-sequence. The intermediate state has native-like secondary but little tertiary structure, and has a compactness between that of the native and unfolded state. Pro-subtilisin folds from the intermediate to the folded state in a single co-operative transition mediated by the pro-sequence. The isolated pro-sequence does not appear from its circular dichroism and 1H-NMR spectrum to have enough intrinsic stabilizing interactions to fold autonomously. However, the difference circular dichroism spectra of the pro-subtilisin variant and native subtilisin suggest that it is folded in the context of the pro-subtilisin molecule. The inability of the pro-subtilisin variant to bind a polypeptide inhibitor supports further the hypothesis that the pro-sequence interacts with subtilisin in the region where the active site is exposed. Our results suggest that the interactions provided by the pro-sequence are important only late on the folding pathway of pro-subtilisin and stabilize the transition state for folding. Kinetic analysis of the refolding reaction in the presence and absence of the pro-sequence reveal this stabilization to be in excess of 7.5 kcal/mol; folding is accelerated more than five orders of magnitude.

Amino Acid Sequence↗

PubMed1993

Engineering a novel specificity in subtilisin BPN'.

The specificity of subtilisin BPN' toward substrates with large hydrophobic P4 residues has been improved by single amino acid replacements at positions 104 and 107. Mutations were designed to (i) increase the size of the P4 binding pocket by replacing Ile107, which is at the bottom of the S4 pocket, by Val, Ala, and Gly and (ii) lose the hydrogen bond between Tyr104 and Ser130 at the entrance of the P4 binding pocket by changing Tyr104 to Phe and thus reduce interactions between small P4 side chains and residue 104. All mutant subtilisins, except for I107V, have increased specificity for residues with large side chains at P4 compared with wild type. Using the conventional definition of specificity as the competition of different substrates for the same enzyme, the I107G mutant subtilisin has one of the largest improvements in substrate specificity reported for subtilisin so far; kcat/KM is increased > 200-fold for Phe compared with Ala as the P4 residue. Further, the activity of I107G toward its specific substrate is comparable to that of the wild-type enzyme. Surprisingly, much of the increase in specificity on mutation of Ile107-->Gly appears to result from a lesion that is transmitted through the structure and effects catalysis. The value of kcat/KM for the small substrate acetyltyrosine ethyl ester, which binds to the S1 pocket, drops by 93% on mutation of Ile107-->Gly. The lesion in subtilisin I107G is complemented, however, on binding of longer substrates that have a large hydrophobic P4 amino acid side chain that can bind in the S4 pocket.

Amino Acid Sequence↗

PubMed1993

Folding of subtilisin BPN': characterization of a folding intermediate.

Subtilisin BPN', an extracellular serine protease from Bacillus amyloliquefaciens, requires a 77 amino acid pro-sequence for correct folding in vivo. We report the observation of a metastable folding intermediate during the refolding of wild-type and a proteolytically inactive mutant subtilisin BPN' that lack the pro-sequence. The addition of the pro-sequence as a separate polypeptide chain results in the folding of the intermediate to the native state. The intermediate state of subtilisin is stable at different temperatures, pH values, and salt concentrations for more than a week and retains its competence for folding. The intermediate state possesses a compactness between that of the native and unfolded states. Although it has native-like secondary structure, it shows no distinct near-UV CD spectrum and has a strongly reduced dispersion in the amide and methyl regions of the 1H NMR spectrum. These indicate considerably less tertiary structure than possessed by the native state. However, the intermediate conformation has regions of stable tertiary structure: it has a high-affinity calcium binding site and, after a first noncooperative transition, unfolds with guanidinium chloride in a cooperative process. These results support a folding mechanism for subtilisin BPN' that comprises a high energy transition state, which is lowered by the interaction with the pro-sequence. The similarity to the folding mechanism of alpha-lytic protease supports the hypothesis that a common folding mechanism has been developed through convergent evolution.

Amino Acid Sequence↗