Core Fold Research Articles

Comparison of the amide proton exchange behavior of the rapidly formed folding intermediate and the native state of an antibody scFv fragment

We have investigated the stability of backbone amide protons of the intermediate and the native state of the scFv fragment of an antibody. Stopped flow experiments analyzed by MS and NMR detected the formation of an exchange protected intermediate within the deadtime of the stopped flow apparatus (17 ms). H/D exchange rates of the native protein identified a number of very stable backbone amide protons in the V L and the V H domains. In the V L domain, this slowly exchanging core of the scFv fragment is similar to the folding core of the intermediate, while the V H domain possesses a great number of very stable amide protons which are not stabilized to a significant degree in the folding intermediate of the scFv fragment.

The P4-P6 domain directs higher order folding of the Tetrahymena ribozyme core.

The active site of group I self-splicing introns occurs at the interface of two proposed structural domains. In the Tetrahymena intron, half of the catalytic core resides within the independently-folding P4-P6 domain while the other half belongs to a putative domain that includes helices P3, P7, P8, and P9 (P3-P9). To determine whether the P3-P9 region of the intron can also fold independently, we used Fe(II)-EDTA and dimethyl sulfate to probe the solvent accessibility of separate fragments of the Tetrahymena intron. These RNAs self-assemble into an active complex in trans, enabling analysis of their structural features both alone and within the complex. Our results show that while the P3-P9 region of the intron retains its secondary structure, most of the tertiary interactions within this region do not form stably in the absence of the P4-P6 domain. This indicates that the P4-P6 domain induces folding in the P3-P9 region, organizing the catalytic cleft between them. Thus the P4-P6 domain provides a scaffold for the folding of the Tetrahymena intron core.

Physical studies of conformational plasticity in a recombinant prion protein.

PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.

The crystal structure of zinc-containing ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7.

The crystal structure of ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 was determined by multiple isomorphous replacement supplemented with anomalous scattering effects of iron atoms in the Fe-S clusters, and refined at 2.0 A resolution to a crystallographic R value of 0.173. The structural model contains a polypeptide chain of 103 amino acid residues, 2 [3Fe-4S] clusters, and 31 water molecules; in this model, the cluster corresponding to cluster II in bacterial dicluster ferredoxins loses the fourth iron atom although it may originally be a [4Fe-4S] cluster. The structure of the archaeal ferredoxin consists of two parts: the core fold part (residues 37-103) and the N-terminal extension part (residues 1-36). The "core fold" part has an overall main-chain folding common to bacterial dicluster ferredoxins, containing two clusters as the active center, two alpha-helices near the clusters, and two sheets of two-stranded antiparallel beta-sheet (the terminal and central beta-sheets). The "N-terminal extension" part is mainly formed by a one-turn alpha-helix and a three-stranded antiparallel beta-sheet. The beta-sheet in the N-terminal extension is hydrogen-bonded with the terminal beta-sheet in the core fold to form a larger beta-sheet. The distinct structural feature of this archaeal ferredoxin lies in the zinc-binding center where the zinc ion is tetrahedrally ligated by four amino acid residues (His 16, His 19, and His 34 from the N-terminal extension, and Asp 76 from the core fold). The zinc ion in the zinc-binding center is located at the interface between the core fold and the N-terminal extension, and connects the beta-sheet in the N-terminal extension and the central beta-sheet in the core fold through the zinc ligation. Thus, the zinc ion plays an important role in stabilizing the structure of the present archaeal ferredoxin by connecting the N-terminal extension and the core fold, which may be common to thermoacidophilic archaeal ferredoxins.

Sequence and structural links between distant ADP-ribosyltransferase families.

The low resolution structure of the Pseudomonas aeroginosa exotoxin A (ETA) presented in 1986 provided the first tantalizing three-dimensional view of an ADP-ribosyl-transferase (ADPRT) catalytic domain. The major features of this protein fold have recurred in the more recently solved crystal structures of the cholera toxin-related heat-labile enterotoxin (LT), diphtheria toxin (DT) and pertussis toxin (PT). A core set of alpha + beta elements define a minimal, conserved scaffold with remarkably plastic sequence requirements-only a single glutamic acid residue critical to catalytic activity is invariant. Other interchangeable residues in locations important for catalysis and binding are suggested by the cocrystal structures of DT with the inhibitor ApUp, ETA with bound AMP and nicotinamide, and DT with substrate NAD-in close accord with labeling and mutagenic data. Faint sequence resemblances that were earlier noticed among prokaryotic ADPRTs have now been securely extended by the structural concordance between toxin folds; more recently, eukaryotic ADPRTs have surfaced and their sequences can be reliably threaded into the conserved core fold. We will briefly summarize efforts in Palo Alto and Hamburg to explore these latter relationships, and to mount a rigorous search for new ADPRT families in the growing sequence databases.

An evaluation of the use of hydrogen exchange at equilibrium to probe intermediates on the protein folding pathway

Methods have been developed recently for probing local fluctuations of protein structure using H/2H-exchange of amide protons at equilibrium. It has been suggested that equilibrium exchange methods can identify the order of events in folding pathways and detect folding cores. We have applied the procedure of measuring the effects of denaturant on the H/2H-exchange of amide protons of barnase, the folding pathway of which is well established. The addition of relatively low concentrations of denaturant causes the mechanism of exchange of amide protons of barnase to change from EX2 to EX1 for the residues that require global unfolding for exchange to occur. This change of mechanism, which would have been missed by some of the standard tests, causes artefacts that could be easily misinterpreted. We also present the thermodynamic argument that measurements at equilibrium cannot give the order of events in folding. Measurement of H/2H-exchange of amide protons at equilibrium, when applied correctly, is an excellent method for analyzing the equilibrium distribution of unfolded and partly folded states. It cannot, in theory and in practice, be used for determining protein folding pathways by itself.

A Tyrosyl-tRNA Synthetase Protein Induces Tertiary Folding of the Group I Intron Catalytic Core

The Neurospora crassamitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns. We have used chemical-structure mapping and footprinting to investigate the interaction of the CYT-18 protein with the N. crassamitochondrial large subunit ribosomal RNA (mt LSU) and ND1introns, which are not detectably self-splicing in vitro. Our results show that both these non-self-splicing introns form most of the short range pairings of the conserved group I intron secondary structure in the absence of CYT-18, but otherwise remain largely unfolded, even at high Mg 2 +concentrations. The binding of CYT-18 promotes the formation of the extended helical domains P6a-P6-P4-P5 (P4-P6 domain) and P8-P3-P7-P9 (P3-P9 domain) and their interaction to form the catalytic core. In iodine-footprinting experiments, CYT-18 binding results in the protection of regions of the phosphodiester backbone expected for tertiary folding of the catalytic core, as well as additional protections that may reflect proximity of the protein. In both introns, most of the putative CYT-18 protection sites are in the P4-P6 domain, the region of the SU intron previously shown to bind CYT-18 as a separate RNA molecule, but additional sites are found in the other major helical domain in P8 and P9 in both introns and in L9 and P7.1/P7.1a in the mt LSU intron. Protease digestion of the CYT-18/intron RNA complexes results in the loss of CYT-18-induced RNA tertiary structure and splicing activity. Considered together with previous studies, our results suggest that CYT-18 binds initially to the P4-P6 region of group I introns to form a scaffold for the assembly of the P3-P9 domain, which may contain additional binding sites for the protein. A three-dimensional model structure of the CYT-18-binding site in group I introns indicates that CYT-18 interacts with the surface of the catalytic core on the side opposite the active-site cleft and may primarily recognize a specific three-dimensional geometry of the phosphodiester backbone of group I introns.

Structure of the Transition State for Folding of a Protein Derived from Experiment and Simulation

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (C/2) are compared in an attempt to derive plausible three-dimensional structural models of the transition state. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the Fvalues. The two are in good agreement overall (correlation coefficient=0.87), which suggests that the theoreticalmodels should provide a structural framework for interpretation of the Fvalues. Both experiment and simulation indicate that the transition state is a distorted form of the native state in which the α-helix is weakened but partially intact and the β-sheet is quite disrupted. As inferred from the Fvalues and observed directly in the simulations, the unfolding of CI2 is cooperative and there is a “folding core” comprising a patch on the α-helix and a portion of the β-sheet, nucleated by interactions between Ala16, Ile49 and other neighbouring residues. The protein becomes less structured radiating away from this core. Overall the data indicate that CI2 folds by a nucleation-collapse mechanism. In the absence of experimental information, we have little confidence that the molecular dynamics simulations are correct, especially when only one or a few simulations are performed. On the other hand, even though the experimentally derived values may reflect the extent of overall structure formation, they do not provide an actual atomic-resolution three-dimensional structure of the transition state. By combining the two approaches, however, we have a framework for interpreting Fvalues and can hopefully arrive at a more trustworthy model of the transition state. The process is in some ways similar to the combination of molecular dynamics and NMR data to solve the tertiary structure of proteins.

The phospho‐β‐galactosidase and synaptotagmin predictions

Two bona fide consensus predictions of secondary and tertiary structure in a protein family, made and announced before experimental structures were known, are evaluated in light of the subsequently determined experimental structures. The first, for phospho-beta-galactosidase, identified the core strands of an 8-fold alpha-beta barrel, and identified the 8-fold alpha-beta barrel itself, which was found in the subsequently determined experimental structure to be the core folding domain. The second, for synaptotagmin, identified seven out of eight beta-strands in the structure correctly, missing only a noncore strand. Three preferred "topologies" were selected from several hundred thousand possible topologies of these seven predicted strands using a rule-based analysis. The subsequently determined experimental structure showed that these seven strands in synaptotagmin adopt one of the three preferred topologies. We were unable, however, to identify the correct topology from among these three topologies.

Is the slow-exchange core the protein folding core?

Is the slow-exchange core the protein folding core?

An empirical energy function for threading protein sequence through the folding motif

In this paper we present a new residue contact potential derived by statistical analysis of protein crystal structures. This gives mean hydrophobic and pairwise contact energies as a function of residue type and distance interval. To test the accuracy of this potential we generate model structures by "threading" different sequences through backbone folding motifs found in the structural data base. We find that conformational energies calculated by summing contact potentials show perfect specificity in matching the correct sequences with each globular folding motif in a 161-protein data set. They also identify correct models with the core folding motifs of hemerythrin and immunoglobulin McPC603 V1-domain, among millions of alternatives possible when we align subsequences with alpha-helices and beta-strands, and allow for variation in the lengths of intervening loops. We suggest that contact potentials reflect important constraints on nonbonded interaction in native proteins, and that "threading" may be useful for structure prediction by recognition of folding motif.