Mechanisms for intragenic complementation at the human argininosuccinate lyase locus.

Argininosuccinate lyase (ASL) is a homotetrameric enzyme that catalyzes the reversible cleavage of argininosuccinate to arginine and fumarate. Deficiencies in the enzyme result in the autosomal, recessive disorder argininosuccinic aciduria. Considerable clinical and genetic heterogeneity is associated with this disorder, which is thought to be a consequence of the extensive intragenic complementation identified in patient strains. Our ability to predict genotype-phenotype relationships is hampered by the current lack of understanding of the mechanisms by which complementation can occur. The 3-dimensional structure of wild-type ASL has enabled us to propose that the complementation between two ASL active site mutant subunits, Q286R and D87G, occurs through a regeneration of functional active sites in the heteromutant protein. We have reconstructed this complementation event, both in vivo and in vitro, using recombinant proteins and have confirmed this hypothesis. The complementation events between Q286R and two nonactive site mutants, M360T and A398D, have also been characterized. The M360T and A398D substitutions have adverse effects on the thermodynamic stability of the protein. Complementation between either the M360T or the A398D mutant and the stable Q286R mutant occurs through the formation of a more stable heteromeric protein with partial recovery of catalytic activity. The detection and characterization of a novel complementation event between the A398D and D87G mutants has shown how complementation in patients with argininosuccinic aciduria may correlate with the clinical phenotype.

Young dyspeptic patients: with a test-and-treat policy, are the benefits of decreased symptom severity and oesophago-gastric-duodenoscopy workload sustained?

OBJECTIVES: To examine the symptom severity and requirement for oesophago-gastro-duodenoscopy (OGD) in young dyspeptic patients 2 years after serological testing for Helicobacter pylori, and to compare the outcomes of seronegative patients with those of seropositive patients. DESIGN: Long-term follow-up study of 232 participants from our previous trial. METHODS: Telephone assessment of patients' symptom severity, scored using a previously validated questionnaire; return of patients for OGD determined using local patient administration system (PAS); and review of medical case notes. RESULTS: Dyspepsia symptom severity of both seronegative and seropositive patients remained reduced compared with initial scores at time of trial recruitment. Symptom severity of seropositive patients was significantly lower than that of seronegative patients (P< 0.001). Seventeen additional patients returned for OGD between six months and two years after the start of the serological study. As 61 of the original 232 study patients returned for OGD within the first six month follow-up period, a total of 78 individuals (34%) had OGD during the two years following the study. Thus, 66% of the original participants avoided OGD. CONCLUSIONS: This study of non-invasive testing for H. pylori in young dyspeptic patients has demonstrated an improvement in symptom severity and a substantial reduction in OGD workload over a 2-year period.

The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold.

Protein W (gpW) from bacteriophage lambda is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis. Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA. Thus, gpW is an intriguing subject for detailed structural studies. We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two alpha-helices and a single two-stranded beta-sheet arranged around a well-packed hydrophobic core. The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution.

Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states.

The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists in equilibrium between folded and unfolded states under non-denaturing buffer conditions. In order to examine the origins of this instability, we have made mutations in the domain and characterized the thermodynamics and kinetics of folding. Results of substitutions of negatively charged residues to neutral amino acid residues suggest that the large electrostatic potential of the domain does not play a dominant role in the instability of the domain. Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwise highly conserved glycine residue in the diverging beta-turn connecting the beta3 and beta4 strands. Mutation of T22 to glycine results in significant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further characterize the basis for the stabilization of the T22 mutant relative to wild-type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native beta-turn structure at this position. Correlation of folding rates with AGADIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, suggests that the free energy of the unfolded state also plays a role in stability. This result highlights the importance of both folded and unfolded states for understanding protein stability.

Role of aromatic residues at the lipid-water interface in micelle-bound bacteriophage M13 major coat protein.

Analyses of transmembrane domains of proteins have revealed that aromatic residues tend to cluster at or near the lipid-water interface of the membrane. To assess protein-membrane interactions of such residues, a viable mutant library was generated of the major coat protein of bacteriophage M13 (a model single membrane-spanning protein) in which one or the other of its interfacial tyrosine residues (Tyr-21 and Tyr-24) is mutated. Using the interfacial tryptophan (Trp-26) as an intrinsic probe, blue shifts in fluorescence emission spectra and quenching constants indicated that mutants with a polar amino acid substitution (such as Y24D or Y24N) are less buried in a deoxycholate micelle environment than in the wild type protein. These polar mutants also exhibited alpha-helix to beta-structure transition temperatures in incremental-heating circular dichroism studies relatively lower than those of wild type and nonpolar mutants (such as Y21V, Y21I, and Y24A), indicating that specific side chains in the lipid-water interface influence local protein-micelle interactions. Mutant Y21F exhibited the highest transition temperature, suggesting that phenylalanine is ostensibly the most effective interfacial anchoring residue. Using phage viability as the assay in a combination of site-directed and saturation mutagenesis experiments, it was further observed that both Tyr residues could not simultaneously be "knocked out". The overall results support the notion that an interfacial Tyr is a primary recognition element for precise strand positioning in vivo, a function that apparently cannot be performed optimally by residues with simple aliphatic character.

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, approximately 20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Analysis of covariation in an SH3 domain sequence alignment: applications in tertiary contact prediction and the design of compensating hydrophobic core substitutions.

We have analyzed sequence covariation in an alignment of 266 non-redundant SH3 domain sequences using chi-squared statistical methods. Artifactual covariations arising from close evolutionary relationships among certain sequence subgroups were eliminated using empirically derived sequence diversity thresholds. This covariation detection method was able to predict residue-residue contacts (side-chain centres of mass within 8 A) in the structure of the SH3 domain with an accuracy of 85 %, which is greater than that achieved in many previous covariation studies. In examining the positions involved most frequently in covariations, we discovered a dramatic over-representation of a subset of five hydrophobic core positions. This covariation information was used to design second and third site substitutions that could compensate for highly destabilizing hydrophobic core substitutions in the Fyn SH3 domain, thus providing experimental data to validate the covariation analysis. The testing of our covariation detection method on 15 other alignments showed that the accuracy of contact prediction is highly variable depending on which sequence alignment is used, and useful levels of prediction accuracy were obtained with only approximately one-third of alignments. The results presented here provide insight into the difficulties inherent in covariation analysis, and suggest that it may have limited usefulness in tertiary structure prediction. On the other hand, our ability to use covariation analysis to design stabilizing combinations of hydrophobic core substitutions attests to its potential utility for gaining deeper insight into the stability determinants and functional mechanisms of proteins with known three-dimensional structures.

Two domains of the epstein-barr virus origin DNA-binding protein, EBNA1, orchestrate sequence-specific DNA binding.

The EBNA1 (for Epstein-Barr nuclear antigen 1) protein of Epstein-Barr virus governs the replication and partitioning of the viral genomes during latent infection by binding to specific recognition sites in the viral origin of DNA replication. The crystal structure of the DNA binding portion of the EBNA1 protein revealed that this region comprises two structural motifs; a core domain, which mediates protein dimerization and is structurally homologous to the DNA binding domain of the papillomavirus E2 protein, and a flanking domain, which mediated all the observed sequence-specific contacts. To test the possibility that the EBNA1 core domain plays a role in sequence-specific DNA binding not revealed in the crystal structure, we examined the effects of point mutations in potential hydrogen bond donors located in an alpha-helix of the EBNA1 core domain whose structural homologue in E2 mediates sequence-specific DNA binding. We show that these mutations severely reduce the affinity of EBNA1 for its recognition site, and that the core domain, when expressed in the absence of the flanking domain, has sequence-specific DNA binding activity. Flanking domain residues were also found to contribute to the DNA binding activity of EBNA1. Thus, both the core and flanking domains of EBNA1 play direct roles in DNA recognition.

Thermodynamic and functional characterization of protein W from bacteriophage lambda. The three C-terminal residues are critical for activity.

Gene product W (gpW), the head-tail joining protein from bacteriophage lambda, provides a fascinating model for studying protein interactions. Composed of only 68 residues, it must interact with at least two other proteins in the phage, and probably with DNA. To study the structural and functional properties of gpW, plasmids were constructed expressing gpW with hexahistidine tag sequences at either the N or C terminus. The purified wild type fusion proteins were found to be stably folded and biologically active. The protein is monomeric as judged by equilibrium ultracentrifugation, and appears to unfold by a cooperative two-state mechanism. Circular dichroism studies indicate that the protein is 47% helical, with a T(m) of 71.3 degrees C, and a DeltaG(u) of 3.01 kcal/mol at 25 degrees C. Mutagenesis of the three hydrophobic C-terminal residues of gpW showed that they are critical for activity, even though they do not contribute to the thermodynamic stability of the protein. Using secondary structure prediction as a guide, we also designed destabilized gpW mutants. The hydrophobic nature of the gpW C terminus caused these mutants to be degraded by the ClpP-containing proteases in Escherichia coli.

Evolutionary conservation in protein folding kinetics.

The sequence and structural conservation of folding transition states have been predicted on theoretical grounds. Using homologous sequence alignments of proteins previously characterized via coupled mutagenesis/kinetics studies, we tested these predictions experimentally. Only one of the six appropriately characterized proteins exhibits a statistically significant correlation between residues' roles in transition state structure and their evolutionary conservation. However, a significant correlation is observed between the contributions of individual sequence positions to the transition state structure across a set of homologous proteins. Thus the structure of the folding transition state ensemble appears to be more highly conserved than the specific interactions that stabilize it.

Day-case percutaneous endoscopic gastrostomy: a viable proposition?

The aim of our study was to evaluate the success rate, complications, and long term outcomes following day-case percutaneous endoscopic gastrostomy (PEG). This retrospective study was carried out in a 650-bed District General hospital in Northamptonshire, UK. Thirty-six patients, aged 28-90 years, were included in the study, 21 males (58%) and 15 females (42%). Indications for PEG insertion included head and neck cancer, dysphagia as a result of primary disease, and AIDS-related malnutrition. Data were collected from the medical and dietetic records. The PEG procedure was successful in 33 patients (92%). In 32 cases (97%) the patient was discharged home. Twenty five of the patients (76%) suffered no complications whilst seven (21%) suffered complications within a month of the procedure. No patient required further surgical intervention. Five patients (15%) died of their primary disease within a month of the procedure. Patients had had their PEG tubes in situ for up to 2.5 years at the end of data collection. We conclude that PEG can be performed as a day-case procedure in stable patients with no increase in complication rate, morbidity, or mortality.

The identification of conserved interactions within the SH3 domain by alignment of sequences and structures.

The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein-protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide-binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side-chain to main-chain hydrogen bonds were observed in the functionally crucial RT-Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved beta-bulge was identified in the second beta-strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold.

The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis.

We have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a Tm of 60 degrees C and a deltaGu of 3.08 kcal/mol. Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild-type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment. Three of the eight mutants constructed in this way displayed increases in Tm ranging from 8 to 15 degrees C with concomitant increases in deltaGu of up to 1.4 kcal/mol. The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a Tm greater than 90 degrees C and a deltaGu more than double that of the wild-type domain. The folding rate of this hyperstable mutant was 10-fold faster than wild-type, while its unfolding rate was fivefold slower. All of the stabilized mutants were still able to bind a target peptide with wild-type affinity. We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies. In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site.

Functional importance of regions in Escherichia coli elongation factor NusA that interact with RNA polymerase, the bacteriophage lambda N protein and RNA.

The association of the essential Escherichia coli protein NusA with RNA polymerase increases pausing and the efficiency of termination at intrinsic terminators. NusA is also part of the phage lambda N protein-modified antitermination complex that functions to prevent transcriptional termination. We have investigated the structure of NusA using various deletion fragments of NusA in a variety of in vitro assays. Sequence and structural alignments have suggested that NusA has both S1 and KH homology regions that are thought to bind RNA. We show here that the portion of NusA containing the S1 and KH homology regions is important for NusA to enhance both termination and antitermination. There are two RNA polymerase-binding regions in NusA, one in the amino-terminal 137 amino acids and the other in the carboxy-terminal 264 amino acids; only the amino-terminal RNA polymerase-binding region provides a functional contact that enhances termination at an intrinsic terminator or antitermination by N. The carboxy-terminal region of NusA is also required for interaction with N and is important for the formation of an N-NusA-nut site or N-NusA-RNA polymerase-nut site complex; the instability of complexes lacking this carboxy-terminal region of NusA that binds N and RNA polymerase can be compensated for by the presence of the additional E. coli elongation factors, NusB, NusG and ribosomal protein S10.

Reducing the endoscopic workload: does serological testing for Helicobacter pylori help?

OBJECTIVE: Serological screening for Helicobacter pylori (H. pylori) in young (< or = 45 years) dyspeptic patients has been used to avoid oesophago-gastro-duodenoscopy (OGD). We used serology to identify seronegative and seropositive patients without sinister symptoms and combined approaches of avoiding OGD in both groups. We aimed to determine the reduction of OGD in this group. DESIGN: Prospective study on the treatment of 232 patients with dyspepsia. SETTING: Six hundred and fifty bed district general hospital serving rural Northamptonshire, UK. INTERVENTIONS: Two hundred and thirty-two patients referred by local general practitioners for OGD were offered serology. Symptom severity was scored using a questionnaire. One hundred and eleven seronegative patients received symptomatic treatment, 105 seropositive patients received triple therapy for 1 week. Sixteen patients with equivocal results were offered OGD. Patients were followed up after 6 months. MAIN OUTCOME MEASURES: Severity of dyspepsia symptoms and proportion of patients returning for OGD. RESULTS: Fifteen equivocal patients underwent OGD, one refused. Forty-six patients (33 seronegative, 13 seropositive) had persisting symptoms and underwent OGD. Mean symptom severity was reduced significantly in equivocal (P<0.01), seronegative (P<0.001) and seropositive (P<0.001) patients. Fewer seronegative patients were symptom-free at follow up compared to seropositive patients (n = 15 (16%) vs n = 48 (51%); P<0.001), 171 patients avoided OGD, a 74% reduction. CONCLUSIONS: Use of H. pylori serology in the management of young dyspeptic patients without sinister symptoms can reduce the OGD workload by 74%, decreasing the length of time that older patients, who are at greater risk of malignant disease, may have to wait for OGD.

A simple in vivo assay for increased protein solubility.

Low solubility is a major stumbling block in the detailed structural and functional characterization of many proteins and isolated protein domains. The production of some proteins in a soluble form may only be possible through alteration of their sequences by mutagenesis. The feasibility of this approach has been demonstrated in a number of cases where amino acid substitutions were shown to increase protein solubility without altering structure or function. However, identifying residues to mutagenize to increase solubility is difficult, especially in the absence of structural knowledge. For this reason, we have developed a method by which soluble mutants of an insoluble protein can be easily distinguished in vivo in Escherichia coli. This method is based on our observation that cells expressing fusions of an insoluble protein to chloramphenicol acetyltransferase (CAT) exhibit decreased resistance to chloramphenicol compared to fusions with soluble proteins. We found that a soluble mutant of an insoluble protein fused to CAT could be selected by plating on high levels of chloramphenicol.

An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S. cerevisiae.

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

Domain exchange experiments in duck delta-crystallins: functional and evolutionary implications.

Delta-crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two delta crystallins, denoted delta1 and delta2. Duck delta2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to delta2/ASL in the amino acid sequence, delta1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras--221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, delta1 or delta2, respectively--were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to delta1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in delta1. The thermodynamic characterization of human ASL (hASL) and duck delta1 and delta2 indicate that delta crystallins are slightly less stable than hASL, with the delta1 being the least stable. The deltaGs of unfolding are 57.25, 63.13, and 70.71 kcal mol(-1) for delta1, delta2, and hASL, respectively. This result was unexpected, and we speculate that delta crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein-protein and protein-solvent interactions.

British Columbia's health reform: "new directions" and accountability.

The health policy New Directions committed the British Columbia government to a population health perspective and extensive community involvement in the health services reform process. The policy envisaged elected citizen boards with authority to raise revenues and exercise a significant degree of local autonomy. Academic and public attention has been paid to the decision in November 1996 to collapse New Directions' two-tier governance structure into a single level. Less attention has been paid to the profound changes that occurred prior to the government's reversal on the question of governance. This paper focuses on those changes. During the critical three years between the 1993 launch of the reform and its formal revision in 1996, the government's positions on elections, taxation power, local autonomy and scope of action for regional boards all changed. Those changes marked a retreat from political accountability to the community and an advance towards managerial accountability to the government.

Mutagenesis of a buried polar interaction in an SH3 domain: sequence conservation provides the best prediction of stability effects.

The SH3 domain from the Fyn tyrosine kinase possesses a buried hydrogen bond between the side chains of a glutamate (Glu24) and a serine (Ser41) residue. Multiple amino acid substitutions were made at these positions to determine the role of this interaction in the stability and conformational specificity of the domain and to assess the relationship between the thermodynamic stability of mutants and sequence conservation seen in the SH3 domain family. Analysis of single and double alanine mutations indicated that the Glu24-Ser41 interaction contributes 0.50 kcal/mol to the stability of the domain. However, disruption of the Glu24-Ser41 interaction did not impair peptide binding function, suggesting that the interaction is not critical for conformational specificity. The stability of the domain was not increased by the replacement of these residues with different combinations of hydrophobic residues or with potential salt bridge forming residues. Despite their similar structural roles in the Fyn SH3 domain, the Ser41 position was considerably more tolerant to substitution than was the Glu24 position. An alignment of >350 different SH3 domains has been completed in our laboratory. A statistically significant correlation was found between the conservation data for the Glu24 and Ser41 positions and the thermodynamic stabilities of the mutants constructed at these positions. Surprisingly, our analysis of sequence alignment data provided a more accurate prediction of the stability of mutants than did examination of the three-dimensional structure of the domain.