NMR-detected order in core residues of denatured bovine pancreatic trypsin inhibitor.

The NMR characteristics of [14-38]Abu, a synthetic variant of BPTI that is partially folded in aqueous buffer near neutral pH, support a model of early folding events which begin with stabilization of the nativelike, slow exchange core [Barbar, E., Hare, M., Daragan, V., Barany, G., and Woodward, C. (1998) Biochemistry 37, 7822-7833 (1)]. In partially folded [14-38]Abu, urea denaturation profiles for representative amide protons show that global unfolding is non-two-state and that core residues require a higher concentration of urea to unfold. Dynamic properties of pH-denatured [14-38]Abu and fully reduced and unfolded BPTI analogue were determined from heteronuclear NMR relaxation measurements at similar solution conditions. Differences at various sites in the polypeptide chain were evaluated from spectral density functions determined from T1, T2, and steady-state heteronuclear NOE data. Although denatured [14-38]Abu contains no persistent secondary structure, its most ordered residues are those that, in native BPTI, fold into the slow exchange core. The fully reduced analogue is significantly more mobile and shows less heterogeneous dynamics, but at 1 degree C, restricted motion is observed for residues in the central segments of the polypeptide chain. These observations indicate that there is a developing core or cores even in highly unfolded species. Apparently the effect of 14-38 disulfide on unfolded

Dimerization and folding of LC8, a highly conserved light chain of cytoplasmic dynein.

Cytoplasmic dynein is a multisubunit ATPase that transforms chemical energy into motion along microtubules. LC8, a 10 kDa light chain subunit of the dynein complex, is highly conserved with 94% sequence identity between Drosophila and human. The precise function of this protein is unknown, but its ubiquitous expression and conservation suggest a critical role in the function of the dynein motor complex. We have overexpressed LC8 from Drosophila melanogaster and characterized its dimerization and folding using analytical ultracentrifugation, size-exclusion chromatography, circular dichroism, and fluorescence spectroscopy. Sedimentation equilibrium measurements of LC8 at pH 7 reveal a reversible monomer-dimer equilibrium with a dissociation constant of 12 microM at 4 degrees C. At lower pH, LC8 dissociates to a monomer, with a transition midpoint at pH 4.8. Far-UV CD and fluorescence spectra demonstrate that pH-dissociated LC8 retains native secondary and tertiary structures, while the diminished near-UV CD signal shows loss of quaternary structure. The observation that dimeric LC8 dissociates at low pH can be explained by titration of a histidine pair in the dimer interface. Equilibrium denaturation experiments with a protein concentration range spanning almost 2 orders of magnitude indicate that unfolding of LC8 dimer is a two-stage process, in which global unfolding is preceded by dissociation to a folded monomer. The nativelike tertiary structure of the monomer suggests a role for the monomer-dimer equilibrium of LC8 in dynein function.

Experimental approaches to protein folding based on the concept of a slow hydrogen exchange core.

In a review of protein hydrogen exchange, we concluded that the slow exchange core is the folding core. By this we mean that the elements of secondary structure carrying the slowest exchanging backbone amides will tend to be the elements of secondary structure to fold first, that partially folded proteins will tend to be most organized in the core, and that peptides made to mimic the slow exchange core will tend to show nativelike structure. These generalizations have led us to ask several experimental questions that will be examined here: (1) In partially folded and unfolded proteins, how do the dynamics and structure of core regions differ from noncore regions? (2) Can we make protein 'core modules' as peptides corresponding to the slow exchange core? Can core modules be covalently linked to make a native state in which one conformation is significantly more stable than all other accessible conformations? (3) In a mutant perturbed outside the core, what are the effects on hydrogen exchange and folding?

Contiguous hydroxyproline residues direct hydroxyproline arabinosylation in Nicotiana tabacum.

Hydroxyproline (Hyp) O-glycosylation characterizes the hydroxyproline-rich glycoprotein (HRGP) superfamily of the plant extracellular matrix. Hyp glycosylation occurs in two modes: Arabinosylation adds short oligoarabinosides (Hyp-arabinosides) while galactosylation leads to the addition of larger arabinogalactan polysaccharides (Hyp-polysaccharides). We hypothesize that sequence-dependent glycosylation of small peptide motifs results in glycomodules. These small functional units in combination with other repetitive peptide modules define the properties of HRGPs. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered noncontiguous Hyp residues. To determine the minimum level of Hyp contiguity that directs arabinosylation, we designed a series of synthetic genes encoding repetitive (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n). A signal sequence targeted these endogenous substrates to the endoplasmic reticulum/Golgi for post-translational proline hydroxylation and glycosylation in transformed Nicotiana tabacum cells. The fusion glycoproteins also contained green fluorescence protein, facilitating their detection and isolation. The (Ser-Pro(2))(n) and (Ser-Hyp(4))(n) fusion glycoproteins yielded Hyp-arabinosides but no Hyp-polysaccharide. The motif (Ser-Pro(3))(n) was incompletely hydroxylated, yielding mixed contiguous/noncontiguous Hyp and a corresponding mixture of Hyp-arabinosides and Hyp-polysaccharides. These results plus circular dichroic spectra of the glycosylated and deglycosylated (Ser-Pro(2))(n), (Ser-Pro(3))(n), and (Ser-Pro(4))(n) modules corroborate the Hyp contiguity hypothesis and indicate that Hyp O-glycosylation is indeed sequence-driven.

Interaction cloning and characterization of RoBPI, a novel protein binding to human Ro ribonucleoproteins.

Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA. RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb RoBPI mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs. RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs. Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs.

NMR characterization of partially folded and unfolded conformational ensembles of proteins.

Studies of unfolded and partially folded proteins provide important insight into the initiation and process of protein folding. This review focuses on the use of nmr in characterization of ensembles of proteins that model the early stages of folding. Analysis of an ensemble includes description of the number of conformations, their structure, relative populations, interconversion rates, and dynamics of subconformations. A chemically synthesized analogue of bovine pancreatic trypsin inhibitor (BPTI), [14-38](Abu), has provided a rare system for characterization of multiple partially folded conformations in slow exchange at near physiological conditions. Multidimensional nmr techniques coupled with selective labeling were used to probe different segments of the polypeptide chain. At each labeled site, there is evidence of slow interconversion between two families of partially folded conformations that in themselves are ensembles of rapidly interconverting conformers. All these conformers display significantly more order in the core relative to the rest of the molecule. For other variants of BPTI that are unfolded at equilibrium, the most ordered structure is also favored in the hydrophobic core residues of the native protein. This is consistent with the hypothesis that the residues that are the first to fold go on to form the most stable, structure-determining part of the protein.

Dynamics of the conformational ensemble of partially folded bovine pancreatic trypsin inhibitor.

A single-disulfide variant of bovine pancreatic trypsin inhibitor (BPTI), [14-38]Abu, is a partially folded ensemble which includes two, and in one case three, conformations that interconvert slowly enough to exhibit separate cross-peaks in the amide region of homonuclear and heteronuclear NMR spectra. Each conformation is itself composed of many subconformations in rapid equilibrium. Partially folded BPTI undergoes local motions that are slow, noncooperative, independent fluctuations of short segments within the chain. Cooperative global unfolding of the ensemble is also observed. Heteronuclear NMR has been used to measure interconversion rate constants of partially folded conformational substates; the rate constants differ for each residue and vary over an order of magnitude. For local fluctuation, the forward rate constants for amide protons of the antiparallel beta-sheet are significantly smaller than the rest of the molecule, consistent with other indications that this is the most stable part of the partially folded protein. The reverse rate constants also vary; they are the highest for Ala 27 in the turn between the strands in the sheet and for Phe 33 in the antiparallel beta-sheet. Global unfolding interconversion rate constants vary over a 3-fold range, consistent with previously observed deviations from two-state behavior. Fast backbone dynamics, from T1, T2, and NOE relaxation parameters, are obtained for the slowly interchanging conformations in the partially folded ensemble. Clear differences are observed between the two conformations; one is more flexible and less compact than the other. In the more flexible and disordered partially folded conformation, intermediate exchange is detected for some backbone amides, namely, those in the central beta-sheet and the turn. These same sheet and turn residues are more ordered in the globally denatured ensemble as well. Our results suggest that the turn initiates formation of a partially folded ensemble in which the slow-exchange core is the most stable region and in which segmental fluctuations reflect multiple nuclei for folding of the rest of the molecule.

Local fluctuations and global unfolding of partially folded BPTI detected by NMR.

The protein [14-38]Abu is a chemically synthesized variant of bovine pancreatic trypsin inhibitor (BPTI) with the 14-38 disulfide bond intact and cysteines 5, 30, 51, and 55 replaced by alpha-amino-n-butyric acid (Abu). At 1-6 degrees C and pH 4.5-6.5, [14-38]Abu is partially folded with a native-like core [1]. Heteronuclear NMR spectra contain two, and in a few cases three or four, exchange cross peaks for each 15N-bound 1H, reporting the presence of two or more conformations that interconvert on a time scale of > or = milliseconds. Thermodynamic analysis of 15N-1H exchange peak volumes as a function of temperature in the range 1-35 degrees C indicates that partially folded [14-38]Abu undergoes local segmental motions as well as cooperative global unfolding. The relative abundance of more folded versus disordered conformations changes throughout the molecule, indicating that various regions of the partially folded protein are disordered to different extents prior to onset of thermal denaturation. This system is unique in providing a measure of the populations of interconverting partially folded conformations, as well as a microscopic view of cooperative folding of a fluctuating ensemble. Although global thermal denaturation is cooperative, significant deviation from simple two-state behavior is reflected in several parameters, including the difference in Tm for thermal unfolding measured by NMR versus circular dichroism.

Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten.

The structural basis of the binding of phenylphosphocholine haptens to antibodies was studied. This was done by preparing antibodies and testing binding to conjugates of phenylphosphocholine. The choice of haptens was made in order to evaluate the contribution of the carrier to binding, and its effect on hapten conformation in the active site. Thus, phosphocholine (PC) was diazophenyl-linked to tyrosine or histidine as single amino acid carriers and to tripeptides or octapeptides containing tyrosine or histidine as central amino acids to which PC was attached. Relative affinity was assessed by inhibition enzyme-linked immunosorbent assay (ELISA) and binding constants were determined by fluorescence quenching. Fluorinated haptens were used to determine the kinetics of binding using 19F nuclear magnetic resonance. The transferred nuclear Overhauser effect was used to characterize conformation of the bound hapten. We had previously shown that nitrophenylphosphocholine unlinked to carrier is bound in the active site as a bent structure [Bruderer, U., Peyton, D. H., Barbar, E., Fellman, J. H., & Rittenberg, M. B. (1992) Biochemistry 31, 584-589]. We show here that this same bent conformation is retained in the active site regardless of the neighboring carrier or the conformation of the hapten in the unbound conjugate. The presence of the carrier residues in the bound state does, however, influence affinity.

Unfolded BPTI variants with a single disulfide bond have diminished non-native structure distant from the crosslink.

BACKGROUND: NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions favored in initial stages of folding, and in early intermediates formed during folding. We have characterized non-random structures favored in unfolded, reduced BPTI [1], and in partially folded BPTI [2]. Here, we report NMR-detected structure of two analogs of unfolded BPTI with one native 14-38 disulfide bond. RESULTS: Analogs Y21A[14-38]Abu and Y23A[14-38]Abu, obtained by chemical synthesis of [14-38]Abu with Y21 or Y23 replaced by alanine, are models for unfolded BPTI with 14-38 the only disulfide. Compared to unfolded BPTI with all three disulfides broken, the unfolded 14-38 BPTI analogs have numerous differences, including loss of non-native, turn-like conformations for beta 2 residues, diminished non-native aromatic-aliphatic NOEs, and increased intermediate chemical exchange of residues that have native-like conformations in partially folded BPTI. Although the Y21A and Y23A analogs have similar CD and NMR properties, specific differences in NOE patterns and in exchange broadening are observed. CONCLUSIONS: Changes in unfolded BPTI associated with formation of the 14-38 disulfide bond are consistent with less non-native structure, and more native-like structure, in residues composing the stable core of antiparallel beta-sheet in partially folded BPTI. Specific differences between Y21A[14-38]Abu and Y23A[14-38]Abu indicate that replacement of Y23 results in less ordered structure than replacement of Y21.

Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor.

Two-dimensional 1H NMR spectra of an analog of reduced BPTI at pH 4.5, 1 degrees C, have been assigned. Spectra indicate considerable conformational averaging, as expected for a flexible, unfolded protein. The presence of extensive nonrandom structure is detected by the presence of NHi-NHi + 1 and aromatic-aliphatic NOEs. Sequential amide-amide NOEs indicate that turn-like conformations are significantly populated at 18 pairs of residues along the chain. Many of these are located in a turn, loop, or helix in native BPTI, but six are observed for contiguous pairs in the segment composed of residues 29-35, which in native BPTI constitute a strand of extended sheet. A novel finding for unfolded proteins is our observation of NOEs implying non-native hydrophobic interactions. Multiple aromatic-aliphatic NOEs are observed for pairs of residues that are within 1-3 residues of each other. Most are non-native and involve residues in both strands of the central antiparallel strand-turn-strand of native BPTI comprised of residues 18-35. All NOEs reported for oligopeptides spanning the BPTI sequence [Kemmink, J., & Creighton, T. (1993) J. Mol. Biol. 234, 861-878] are observed in reduced BPTI, but many others are present as well. Similar spectra are obtained for naturally occurring BPTI reduced by dithiothreitol, BPTI with cysteines replaced by alpha-amino-n-butyric acid, and BPTI mutant F45A reduced by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic structure of a highly ordered beta-sheet molten globule: multiple conformations with a stable core.

The structure of [14-38]Abu, a variant of bovine pancreatic trypsin inhibitor (BPTI) with only the 14-38 disulfide bridge intact, has been analyzed by two-dimensional 1H and 1H-15N NMR. Except for the 18-24, 29-35 antiparallel beta-sheet, residues in all regions of the molecule give two exchange cross peaks for each 1H; for one residue, Gly 37, three exchange cross peaks are observed. The presence of exchange cross peaks indicates that the residues sample conformations that interconvert on a time scale of milliseconds or longer. Over 90% of the NMR spectra have been assigned, including backbone and side chain atoms and their exchange cross peaks. Analyses of chemical shifts, chemical exchange, hydrogen isotope exchange, and NOEs indicate that [14-38]Abu at pH 4.5 and 1 degree C is an ensemble of interconverting conformations, in all of which the 18-24, 29-35 antiparallel beta-sheet is native-like and intact. Outside the antiparallel beta-sheet, residues undergo local order/disorder transitions. The stable structure of [14-38]Abu is not in the vicinity of the 14-38 disulfide bond but rather is in the slow-exchange core. NOE analysis indicates that the main tertiary interactions involve hydrophobic contacts with the rings of Tyr 21, Tyr 23, and Tyr 35. As a model for early folding intermediates, the structure of [14-38]Abu suggests that BPTI folding is initiated by stabilization of a turn existing in the unfolded protein and involves both local and nonlocal hydrophobic interactions.

Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa > 9.

Apparent pKa values of active site residues Asp26, Cys32, and Cys35 in reduced thioredoxin have been characterized. Both wild-type thioredoxin and mutant D26A thioredoxin were selectively 13C-enriched on cysteine beta-carbons. In both proteins, the variation with pH of 1HB1, 1HB2, and 13CB NMR chemical shifts has been measured. In wild-type reduced thioredoxin, for both cysteines, the pH versus chemical shift plots of HB1 protons can be fit to one titration with pKa values of 7.0-7.1. In contrast, the HB2 protons and beta-carbons give pH--chemical shift plots that clearly reflect more than one titration; fits to the data give apparent pKa values of 7.0-7.3 and 9.5 for HB2 protons and 7.5-7.9 and 9.2-10.2 for CB carbons. In reduced D26A, all three probe chemical shifts have a pH dependence that is fit by one titration with pKa of 7.4-7.9. The absence of a titration with pKa > 9 in D26A, taken together with cysteine thiol pKa values of 7.1 and 7.9 determined by Raman spectroscopy [Li et al. (1993) Biochemistry 32, 5800-5808], indicates that the pKa > 9 in reduced thioredoxin is that of Asp26. This is highly significant in view of the previous observation that, in oxidized thioredoxin, Asp26 pKa is 7.5 [Langsetmo et al. (1991) Biochemistry 30, 7603-7609]. The very high pKa values of these carboxyls is consistent with their local environment in the three-dimensional structure; the Asp26 side chain in oxidized thioredoxin is almost but not completely buried, and in reduced thioredoxin it may be even more buried.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR behavior of the aromatic protons of bovine neurophysin-I and its peptide complexes: implications for solution structure and for function.

The NMR behavior of the aromatic protons of bovine neurophysin-I and its complexes was interpreted with reference to the 2.8 A crystal structure of the dipeptide complex of bovine neurophysin-II and to mechanisms underlying the thermodynamic linkage between neurophysin dimerization and peptide binding. Large binding-induced shifts in the ring proton signals of Tyr-2 of ligand peptides (approximately 0.5 ppm upfield and approximately 0.35 ppm downfield at 25 degrees C for the 3,5- and 2,6-ring protons, respectively) were demonstrated. Consistent with the crystal structure, and in disagreement with conclusions by other investigators, evidence is presented indicating the absence of dipolar contact between Tyr-2 ring protons and protein Phe ring protons. The large binding-induced shifts are attributed to a strong influence of proximal neurophysin carbonyl and disulfide groups on the bound Tyr-2 ring, of potential importance in binding specificity. Resolution of the behavior of neurophysin Phe residues -22 and -35 and of their proton NOE contacts provided insights into the conformational changes associated with peptide binding and with dimerization. Within the amino domain of the protein, as evidenced by the behavior of interface residue Phe-35 and its NOE contacts, binding-induced changes in the subunit interface appeared to involve principally the junction between this interface region and the 3,10-helix that connects it to the binding site in the liganded state. By contrast, as judged by the NOE contacts of His-80, the corresponding interface participant of the carboxyl domain, peptide binding induced a marked decrease in side-chain mobility within the carboxyl domain segment of the interface. Interactions of Phe-22 with protons assigned to Ala-68, neither of which is an interface participant, were demonstrated to be markedly altered both by dimerization in the unliganded state and by peptide binding to the dimer. Since Phe-22 is adjacent to the peptide-binding site, the results collectively support a model in which conformational differences between unliganded monomer and dimer are important contributors to the preferential binding of peptide to the dimer and indicate that the amino and carboxyl domain segments of the interface, which are homologous, are affected differently by peptide binding.

Unfolded BPTI variants with a single disulfide bond have diminished non-native structure distant from the crosslink.

Backgound. NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions favored in initial stages of folding, and in early intermediates formed during folding. We have characterized non-random structures favored in unfolded, reduced BPTI [1], and in partially folded BPTI [2]. Here, we report NMR-detected structure of two analogs of unfolded BPTI with one native 14-38 disulfide bond. Results. Analogs Y21A[14-38]Abu and Y23A[14-38]Abu, obtained by chemical synthesis of [14-38]Abu with Y21 or Y23 replaced by alanine, are models for unfolded BPTI with 14-38 the only disulfide. Compared to unfolded BPTI with all three disulfides broken, the unfolded 14-38 BPTI analogs have numerous differences, including loss of non-native, turn-like conformations for beta2 residues, diminished non-native aromatic-aliphatic NOEs, and increased intermediate chemical exchange of residues that have native-like conformations in partially folded BPTI. Although the Y21A and Y23A analogs have similar CD and NMR properties, specific differences in NOE patterns and in exchange broadening are observed. Conclusion. Changes in unfolded BPTI associated with formation of the 14-38 disulfide bond are consistent with less non-native structure, and more native-like structure, in residues composing the stable core of antiparallel beta-sheet in partially folded BPTI. Specific differences between Y21A[14-38]Abu and Y23A[14-38]Abu indicate that replacement of Y23 results in less ordered structure than replacement of Y21.

Hapten conformation in the combining site of antibodies that bind phenylphosphocholine.

We have shown previously that anti-phenylphosphocholine antibodies elicited by phosphocholine-keyhole limpet hemocyanin can be divided into two populations according to their ability to recognize the two hapten analogues p-nitrophenylphosphocholine (NPPC) and p-nitrophenyl 3,3-dimethylbutyl phosphate (NPDBP). These analogues differ from each other in that NPPC has a positively charged nitrogen in the choline moiety, whereas NPDBP lacks the positively charged nitrogen. Group II-A antibodies bind only NPPC, whereas group II-B antibodies bind both ligands. Here, by infrared and nuclear magnetic resonance spectroscopic investigations, we find that when free in solution NPPC has a predominantly fixed structure in which the termini approach each other, probably due to electrostatic interactions within the molecule; this "bent" structural feature is retained when the ligand is bound by antibody. In contrast, the structure of unbound NPDBP is less fixed, being characterized by rapidly interchanging conformations corresponding to an open chain structure with less overall proximity of the termini compared to NPPC. The overall shape of NPPC is essentially unaltered by binding, whereas in the case of NPDBP what was a minor conformation in the unbound state becomes the predominate conformation of the bound ligand. Thus, our results are consistent with these antibodies providing a molecular template for stabilizing the conformation of the bound ligand.