High prevalence of K-ras-2 mutations in hepatocellular carcinomas in workers exposed to vinyl chloride.

OBJECTIVES: Vinyl chloride (VC) and its metabolites are human carcinogens associated with liver angiosarcomas (LAS) and also with hepatocellular carcinomas (HCCs). In VC associated LAS mutations of the K-ras-2 gene have been reported, however, no data about the prevalence of such mutations in VC-associated HCCs are available. The aim of the study was to evaluate possible specific K-ras-2 oncogene mutations in the case of HCCs due to VC. METHODS: The presence of K-ras-2 mutations was analysed in tissue from 12 patients with VC-associated HCCs. All patients had known long-term exposure to VC (average exposure amount: 9,942 ppm-years). Twenty patients with hepatocellular carcinoma due to hepatitis B (n = 7), hepatitis C (n = 5) and alcoholic liver cirrhosis (n= 8) served as a control group. The specific mutations were determined by direct sequencing of codons 12 and 13 of the K-ras-2 gene in carcinomatous and adjacent non-neoplastic liver tissue after microdissection. Immunohistochemical analysis was performed to detect p21ras protein. RESULTS: K-ras-2 mutations were found in five of 12 (42%) examined HCCs and in three cases of adjacent non-neoplastic liver tissue (25%). There were three guanine to adenine (G --> A) point mutations in the tumour tissue. All three mutations found in non-neoplastic liver from VC-exposed patients were also G --> A point mutations (codon 12- and codon 13-aspartate mutations). Within the control group, K-ras-2 mutations were found in three of 20 (15%) examined HCCs. CONCLUSIONS: Mutations of the K-ras-2 gene in hepatocellular carcinomas associated with VC exposure are frequent events. We observed a K-ras-2 mutation pattern characteristic of chloroethylene oxide, one of the carcinogenic metabolites of VC analysed in animal models. Our results suggest that VC had direct toxic effects not only on endothelial cells but also on hepatocytes, as it was previously only described in animal models.

Backbone and side chain dynamics of uncomplexed human adipocyte and muscle fatty acid-binding proteins.

Adipocyte lipid-binding protein (A-LBP) and muscle fatty acid-binding protein (M-FABP) are members of a family of small ( approximately 15 kDa) cytosolic proteins that are involved in the metabolism of fatty acids and other lipid-soluble molecules. Although highly homologous (65%) and structurally very similar, A-LBP and M-FABP display distinct ligand binding characteristics. Since ligand binding may be influenced by intrinsic protein dynamical properties, we have characterized the backbone and side chain dynamics of uncomplexed (apo) human A-LBP and M-FABP. Backbone dynamics were characterized by measurements of 15N T1 and T2 values and ¿1H¿-15N NOEs. These data were analyzed using model-free spectral density functions and reduced spectral density mapping. The dynamics of methyl-containing side chains were charaterized by measurements of 2H T1 and T1rho relaxation times of 13C1H22H groups. The 2H relaxation data were analyzed using the model-free approach. For A-LBP, 15N relaxation data were obtained for 111 residues and 2H relaxation data were obtained for 42 methyl groups. For M-FABP, 15N relaxation data were obtained for 111 residues and 2H relaxation data were obtained for 53 methyl groups. The intrinsic flexibilities of these two proteins are compared, with particular emphasis placed on binding pocket residues. There are a number of distinct dynamical differences among corresponding residues between the two proteins. In particular, many residues display greater backbone picosecond to nanosecond and/or microsecond to millisecond time scale mobility in A-LBP relative to M-FABP, including F57, K58, and most residues in alpha-helix 2 (residues 28-35). Variations in the dynamics of this region may play a role in ligand selectivity. The side chains lining the fatty acid binding pocket display a wide range of motional restriction in both proteins. Side chains showing distinct dynamical differences between the two proteins include those of residues 20, 29, and 51. This information provides a necessary benchmark for determining dynamical changes induced by ligand binding and may ultimately lead to an enhanced understanding of ligand affinity and selectivity among fatty acid-binding proteins.

Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes.

Backbone-atom resonances have been assigned for both the substrate-free and the NADP+-complexed forms of UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a monomeric, 347-residue (38.5 kDa) flavoenzyme essential for bacterial cell-wall biosynthesis. NMR studies were performed using perdeuterated, uniformly 13C/15N-labeled samples of MurB. In the case of substrate-free MurB, one or more backbone atoms have been assigned for 334 residues (96%). The assigned backbone atoms include 309 1HN and 15N atoms (94%), 315 13CO atoms (91%), 331 13C(alpha) atoms (95%), and 297 13C(beta) atoms (93%). For NADP+-complexed MurB, one or more backbone atoms have been assigned for 313 residues (90%); these include 283 1HN and 15N atoms (86%), 305 13CO atoms (88%), 310 13C(alpha) atoms (89%), and 269 13C(beta) atoms (84%). The strategies used for obtaining resonance assignments are described in detail. Information on the secondary structure in solution for both the substrate-free and NADP+-complexed forms of the enzyme has been derived both from 13C(alpha) and 13C(beta) chemical-shift deviations from random-coil values and from 1HN-1HN NOEs. These data are compared to X-ray crystallographic structures of substrate-free MurB and MurB complexed with the UDP-N-acetylglucosamine enolpyruvate (UNAGEP) substrate. NADP+ binding induces significant chemical-shift changes in residues both within the known UNAGEP and FAD binding pockets and within regions known to undergo conformational changes upon UNAGEP binding. The NMR data indicate that NADP+ and UNAGEP utilize the same binding pocket and, furthermore, that the binding of NADP+ induces structural changes in MurB. Finally, many of the residues within the UNAGEP/NADP+ binding pocket were difficult to assign due to dynamic processes which weaken and/or broaden the respective resonances. Overall, our results are consistent with MurB having a flexible active site.

Solution structure of the Grb2 N-terminal SH3 domain complexed with a ten-residue peptide derived from SOS: direct refinement against NOEs, J-couplings and 1H and 13C chemical shifts.

Refined ensembles of solution structures have been calculated for the N-terminal SH3 domain of Grb2 (N-SH3) complexed with the ac-VPPPVPPRRR-nh2 peptide derived from residues 1135 to 1144 of the mouse SOS-1 sequence. NMR spectra obtained from different combinations of both 13C-15N-labeled and unlabeled N-SH3 and SOS peptide fragment were used to obtain stereo-assignments for pro-chiral groups of the peptide, angle restraints via heteronuclear coupling constants, and complete 1H, 13C, and 15N resonance assignments for both molecules. One ensemble of structures was calculated using conventional methods while a second ensemble was generated by including additional direct refinements against both 1H and 13C(alpha)/13C(beta) chemical shifts. In both ensembles, the protein:peptide interface is highly resolved, reflecting the inclusion of 110 inter-molecular nuclear Overhauser enhancement (NOE) distance restraints. The first and second peptide-binding sub-sites of N-SH3 interact with structurally well-defined portions of the peptide. These interactions include hydrogen bonds and extensive hydrophobic contacts. In the third highly acidic sub-site, the conformation of the peptide Arg8 side-chain is partially ordered by a set of NOE restraints to the Trp36 ring protons. Overall, several lines of evidence point to dynamical averaging of peptide and N-SH3 side-chain conformations in the third subsite. These conformations are characterized by transient charge stabilized hydrogen bond interactions between the peptide arginine side-chain hydrogen bond donors and either single, or possibly multiple, acceptor(s) in the third peptide-binding sub-site.

Orientation of peptide fragments from Sos proteins bound to the N-terminal SH3 domain of Grb2 determined by NMR spectroscopy.

NMR spectroscopy has been used to characterize the protein-protein interactions between the mouse Grb2 (mGrb2) N-terminal SH3 domain complexed with a 15-residue peptide (SPLLPKLPP-KTYKRE) corresponding to residues 1264-1278 of the mouse Sos-2 (mSos-2) protein. Intermolecular interactions between the peptide and 13C-15N-labeled SH3 domain were identified in half-reverse-filtered 2D and 3D NOESY experiments. Assignments for the protons involved in interactions between the peptide and the SH3 domain were confirmed in a series of NOESY experiments using a set of peptides in which different leucine positions were fully deuterated. The peptide ligand-binding site of the mGrb2 N-terminal SH3 domain is defined by the side chains of specific aromatic residues (Tyr7, Phe9, Trp36, Tyr52) that form two hydrophobic subsites contacting the side chains of the peptide Leu4 and Leu7 residues. An adjacent negatively charged subsite on the SH3 surface is likely to interact with the side chain of a basic residue at peptide position 10 that we show to be involved in binding. The peptide-binding site of the SH3 is characterized by large perturbations of amide chemical shifts when the peptide is added to the SH3 domain. The mGrb2 N-terminal SH3 domain structure in the complex is well-defined (backbone RMSD of 0.56 +/- 0.21 calculated over the backbone N, C alpha, and C atoms of residues 1-54). The structure of the peptide in the complex is less well-defined but displays a distinct orientation.(ABSTRACT TRUNCATED AT 250 WORDS)

An automated procedure for the assignment of protein 1HN, 15N, 13C alpha, 1H alpha, 13C beta and 1H beta resonances.

A computer algorithm that determines the 1HN, 15N, 13C alpha, 1H alpha, 13C beta and 1H beta chemical-shift assignments of protein residues with minimal human intervention is described. The algorithm is implemented as a suite of macros that run under a modified version of the FELIX 1.0 program (Hare Research, Bothell, WA). The input to the algorithm is obtained from six multidimensional, triple-resonance experiments: 3D HNCACB, 3D CBCA(CO)HN, 4D HNCAHA, 4D HN(CO)CAHA, 3D HBHA(CO)NH and 3D HNHA(Gly). For small proteins, the two 4D spectra can be replaced by either the 3D HN(CA)HA, 3D H(CA)NNH, or the 15N-edited TOCSY-HSQC experiments. The algorithm begins by identifying and collecting the intraresidue and sequential resonances of the backbone and 13C beta atoms into groups. These groups are sequentially linked and then assigned to residues by matching the 13C alpha and 13C beta chemical-shift profiles of the linked groups to that of the protein's primary structure. A major strength of the algorithm is its ability to overcome imperfect data, e.g., missing or overlapping peaks. The viability of the procedure is demonstrated with two test cases. In the first, NMR data from the six experiments listed above were used to reassign the backbone resonances of the 93-residue human hnRNP C RNA-binding domain. In the second, a simulated cross-peak list, generated from the published NMR assignments of calmodulin, was used to test the ability of the algorithm to assign the backbone resonances of proteins containing internally homologous segments. Finally, the automated method was used to assign the backbone resonances of apokedarcidin, a previously unassigned, 114-residue protein.

Sequential 1H, 13C, and 15N NMR assignments and solution conformation of apokedarcidin.

Kedarcidin is a recently discovered antitumor antibiotic chromoprotein. The solution conformation of the kedarcidin apoprotein (114 residues) has been characterized by heteronuclear multidimensional NMR spectroscopy. Sequence-specific backbone atom resonance assignments were obtained for a uniformly 13C/15N-enriched sample of apokedarcidin via a semiautomated analysis of 3D HNCACB, 3D CBCA-(CO)NH, 4D HNCAHA, 4D HN(CO)CAHA, 3D HBHA(CO)NH, and 3D HNHA(Gly) spectra. Side-chain assignments were subsequently obtained by analysis of (primarily) 3D HCCH-TOCSY and HCCH-COSY spectra. A qualitative analysis of the secondary structure is presented on the basis of 3J alpha NH coupling constants, deviations of 13C alpha and 13C beta chemical shifts from random coil values, and NOEs observed in 3D 15N- and 13C-edited NOESY-HSQC spectra. This analysis revealed a four-stranded antiparallel beta-sheet, a three-stranded antiparallel beta-sheet, and two two-standed antiparallel beta-sheets. The assignments of cross-peaks in the 3D NOESY spectra were assisted by reference to a preliminary model of apokedarcidin built using the program CONGEN starting from the X-ray structure of the homologous protein aponeocarzinostatin. An ensemble of 15 apokedarcidin solution structures has been generated by variable target function minimization (DIANA program) and refined by simulated annealing (X-PLOR program). The average backbone atom root-mean-square difference between the individual structures and the mean coordinates is 0.68 +/- 0.08 A. The overall fold of apokedarcidin is well-defined; it is composed of an immunoglobulin-like seven-stranded antiparallel beta-barrel and a subdomain containing two antiparallel beta-ribbons. Highly similar tertiary structures have been previously reported for the related proteins neocarzinostatin, macromomycin, and actinoxanthin. Important structural features are revealed, including the dimensions of the chromophore-binding pocket and the locations of side chains that are likely to be involved in chromophore stabilization.

Solution structure of an isolated antibody VL domain.

The solution structure of the isolated VL domain of the anti-digoxin antibody 26-10 has been determined using data derived from heteronuclear multi-dimensional nuclear magnetic resonance (n.m.r.) experiments. Analytical ultracentrifugation and n.m.r. data demonstrate that the VL domain is only weakly associating (Kd = 2.5 (+/- 0.7) mM) and that it experiences a rapid monomer/dimer equilibrium under the n.m.r. experimental conditions. Therefore, the results reported here represent the first structure determination of an antibody VL domain in the absence of fixed quaternary interactions. The structure determination is based on 930 proton-proton distance constraints, 113 dihedral angle constraints, and 46 hydrogen bond constraints. Eighty initial structures were calculated with the variable target function program DIANA; of these, 31 were accepted on the basis of satisfaction of constraints (no distance constraint violations > 0.5 A; target function < 3.0 A2). Accepted DIANA structures were refined by restrained energy minimization using the X-PLOR program. The 15 best energy-minimized DIANA structures were chosen as a representative ensemble of solution conformations. The average root-mean-square differences (r.m.s.d.) between the individual structures of this ensemble and the mean coordinates is 0.85 (+/- 0.10) A for all backbone atoms and 1.29 (+/- 0.10) A for all heavy atoms. For beta-strands A, B, C, D, E and F, the average backbone atom r.m.s.d. to the mean structure is 0.46 (+/- 0.06) A. A higher-resolution ensemble, with all backbone atom and all heavy atom r.m.s.d.s. to the mean coordinates of 0.54 (+/- 0.08) A and 0.98 (+/- 0.12) A, respectively, was obtained by X-PLOR simulated annealing refinement of the 15 energy-minimized DIANA structures. A detailed analysis of the original ensemble of 15 energy-minimized DIANA structures is presented, as this ensemble retains a broader, and possibly more realistic, sampling of conformation space. The backbone atom and all heavy atom r.m.s.d.s between the mean energy-minimized DIANA structure and the X-ray derived coordinates of the VL domain within the Fab/digoxin complex are 1.05 A and 1.56 A, respectively. Subtle differences between the solution and X-ray structures occur primarily in CDR2, CDR3, beta-strands A, F and G, and localized regions of hydrophobic packing. Overall, these results demonstrate that the 26-10 VL domain conformation is determined primarily by intradomain interactions, and that quaternary VL-VH association induces relatively minor conformational adjustments.

Production and characterization of an antibody Fv fragment 15N-labeled in the VL domain only.

Procedures for separating and recombining the light-chain variable (VL) and heavy-chain variable (VH) domains of antibody Fv fragments have been applied to produce a recombinant Fv of the anti-digoxin antibody 26-10 that is 15N-labeled exclusively in the VL domain. Comparison of a two-dimensional 1H-15N heteronuclear single-quantum correlation (HSQC) spectrum of the reconstituted Fv with a HSQC spectrum of a fully 15N-labeled Fv sample reveals that all 1H-15N correlations of the VL domain align precisely in both spectra. Assignments for 105 of the 106 backbone HN groups of the VL domain within the reconstituted Fv have been obtained by analysis of three-dimensional nuclear Overhauser effect spectroscopy-HSCQ and total correlation spectroscopy-HSQC spectra with reference to assignments previously reported for the isolated VL domain. Chemical shift differences between the isolated VL domain and the VL domain within the Fv are moderately correlated with proximity to the surfaces of the VH domain and bound hapten (ouabain) as defined by X-ray crystallography and molecular modeling. These results demonstrate that nuclear magnetic resonance studies of reconstituted antibody Fv fragments, in conjunction with investigations of isolated antibody domains, can yield extensive resonance assignments for the Fv. This will facilitate detailed studies of antigen-antibody and domain-domain interactions.

Aliphatic 1H and 13C resonance assignments for the 26-10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy.

Extensive 1H and 13C assignments have been obtained for the aliphatic resonances of a uniformly 13C- and 15N-labeled recombinant VL domain from the anti-digoxin antibody 26-10. Four-dimensional triple resonance NMR data acquired with the HNCAHA and HN(CO)CAHA pulse sequences [Kay et al. (1992) J. Magn. Reson., 98, 443-450] afforded assignments for the backbone HN, N, H alpha and C alpha resonances. These data confirm and extend HN, N and H alpha assignments derived previously from three-dimensional 1H-15N NMR studies of uniformly 15N-labeled VL domain [Constantine et al. (1992), Biochemistry, 31, 5033-5043]. The identified H alpha and C alpha resonances provided a starting point for assigning the side-chain aliphatic 1H and 13C resonances using three-dimensional HCCH-COSY and HCCH-TOCSY experiments [Clore et al. (1990), Biochemistry, 29, 8172-8184]. The C alpha and C beta chemical shifts are correlated with the VL domain secondary structure. The extensive set of side-chain assignments obtained will allow a detailed comparison to be made between the solution structure of the isolated VL domain and the X-ray structure of the VL domain within the 26-10 Fab.

Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two-dimensional NMR spectroscopy.

The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.

Production of stable anti-digoxin Fv in Escherichia coli.

We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.

Interaction of the RNA-binding domain of the hnRNP C proteins with RNA.

The hnRNP C proteins are among the most abundant and avid pre-mRNA-binding proteins and they contain a consensus sequence RNA-binding domain (RBD) that is found in a large number of RNA-binding proteins. The interaction of the RBD of the hnRNP C proteins with an RNA oligonucleotide [r(U)8] was monitored by nuclear magnetic resonance (NMR). 15N and 13C/15N-labelled hnRNP C protein RBD was mixed with r(U)8 and one- and two-dimensional (1D and 2D) NMR spectra were recorded in a titration experiment. NMR studies of the uncomplexed 93 amino acid hnRNP C RBD (Wittekind et al., 1992) have shown that it has a compact folded structure (beta alpha beta beta alpha beta), which is typical for the RBD of this family of proteins and which is comprised of a four-stranded antiparallel beta-sheet, two alpha-helices and relatively unstructured amino- and carboxy-terminal regions. Sequential assignments of the polypeptide main-chain atoms of the hnRNP C RBD-r(U)8 complex revealed that these typical structural features are maintained in the complex, but significant perturbations of the chemical shifts of amide group atoms occur in a large number of residues. Most of these residues are in the beta-sheet region and especially in the terminal regions of the RBD. In contrast; chemical shifts of the residues of the well conserved alpha-helices, with the exception of Lys30, are not significantly perturbed. These observations localize the candidate residues of the RBD that are involved in the interaction with the RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

1H, 13C, and 15N NMR assignments and global folding pattern of the RNA-binding domain of the human hnRNP C proteins.

The hnRNP C1 and C2 proteins are abundant nuclear proteins that bind avidly to heterogeneous nuclear RNAs (hnRNAs) and appear to be involved with pre-mRNA processing. The RNA-binding activity of the hnRNP C proteins is contained in the amino-terminal 94 amino acid RNA-binding domain (RBD) that is identical for these two proteins. We have obtained the 1H, 13C, and 15N NMR assignments for the RBD of the human hnRNP C proteins. The assignment process was facilitated by extensive utilization of three- and four-dimensional heteronuclear-edited spectra. Sequential assignments of the backbone resonances were made using a combination of 15N-edited 3D NOESY-HMQC, 3D TOCSY-HMQC, and 3D TOCSY-NOESY-HSQC as well as 3D HNCA, HNCO, and HCACO spectra. Side-chain resonances were assigned using 3D HCCH-COSY and 3D HCH-TOCSY spectra. Four-dimensional 13C/13C-edited NOESY and 13C/15N-edited NOESY experiments were used to unambigously resolve NOEs. The overall global folding pattern was established by calculating a set of preliminary structures using constraints derived from the sequential NOEs and a small number of long-range NOEs. The beta alpha beta-beta alpha beta domain structure exhibits an antiparallel beta-sheet with the conserved RNP 1 and RNP 2 sequences [Dreyfuss et al. (1988) Trends Biochem. Sci. 13, 86-91] located adjacent to one another as the two inner strands of the beta-sheet.

Sequential 1H and 15N NMR assignments and secondary structure of a recombinant anti-digoxin antibody VL domain.

A uniformly 15N-labeled recombinant light-chain variable (VL) domain from the anti-digoxin antibody 26-10 has been investigated by heteronuclear two-dimensional (2D) and three-dimensional (3D) NMR spectroscopy. Complementary homonuclear 2D NMR studies of the unlabeled VL domain were also performed. Sequence-specific assignments for 97% of the main-chain and 70% of the side-chain proton resonances have been obtained. Patterns of nuclear Overhauser effects observed in 2D NOESY, 3D NOESY-HSQC, and 3D NOESY-TOCSY-HSQC spectra afford a detailed characterization of the VL domain secondary structure in solution. The observed secondary structure--a nine-stranded antiparallel beta-barrel--corresponds to that observed crystallographically for VL domains involved in quaternary associations. The locations of slowly exchanging amide protons have been discerned from a 2D TOCSY spectrum recorded after dissolving the protein in 2H2O. Strands B, C, E, and F are found to be particularly stable. The possible consequences of these results for domain-domain interactions are discussed.

A refocused and optimized HNCA: increased sensitivity and resolution in large macromolecules.

A 3D optimized, refocused HNCA experiment is described. It is demonstrated to yield a dramatic increase in sensitivity when applied to [13C, 15N]-labeled human carbonic anhydrase II, a 29-kDa protein. The reasons for the gain in sensitivity are discussed, and 3 distinct areas for further development are indicated.

Sequence-specific 1H NMR resonance assignments of Bacillus subtilis HPr: use of spectra obtained from mutants to resolve spectral overlap.

On the basis of an analysis of two-dimensional 1H NMR spectra, the complete sequence-specific 1H NMR assignments are presented for the phosphocarrier protein HPr from the Gram-positive bacterium Bacillus subtilis. During the assignment procedure, extensive use was made of spectra obtained from point mutants of HPr in order to resolve spectral overlap and to provide verification of assignments. Regions of regular secondary structure were identified by characteristic patterns of sequential backbone proton NOEs and slowly exchanging amide protons. B. subtilis HPr contains four beta-strands that form a single antiparallel beta-sheet and two well-defined alpha-helices. There are two stretches of extended backbone structure, one of which contains the active site His15. The overall fold of the protein is very similar to that of Escherichia coli HPr determined by NMR studies [Klevit, R. E., & Waygood, E. B. (1986) Biochemistry 25, 7774-7781].

Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).

Common structural changes accompany the functional inactivation of HPr by seryl phosphorylation or by serine to aspartate substitution.

Although many proteins are known to be regulated via reversible phosphorylation, little is known about the mechanism by which the covalent modification of seryl, threonyl, or tyrosyl residues alters the activities of the target systems. To address this question, modified versions of Bacillus subtilus HPr, a protein component of the bacterial phosphotransferase system, have been studied by 1H NMR spectroscopy. Phosphorylation at Ser46 or a Ser to Asp substitution at this position inactivates HPr [Reizer, J., Sutrina, S. L., Saier, M. H., Stewart, G. C., Peterkofsky, A., & Reddy, P. (1989) EMBO J. 8, 2111-2120]. Two-dimensional spectra of these two modified proteins display nearly identical proton chemical shifts that differ significantly from those observed in the spectra of the unphosphorylated, wild-type protein and of functionally active HPr mutants. The results demonstrate that the functional inactivation of HPr brought about by the serine to aspartate mutation is accompanied by the same structural changes that occur when HPr is phosphorylated at Ser46.