Disruptive NADSYN1 Variants Implicated in Congenital Vertebral Malformations.

Genetic perturbations in nicotinamide adenine dinucleotide de novo (NAD) synthesis pathway predispose individuals to congenital birth defects. The NADSYN1 encodes the final enzyme in the de novo NAD synthesis pathway and, therefore, plays an important role in NAD metabolism and organ embryogenesis. Biallelic mutations in the NADSYN1 gene have been reported to be causative of congenital organ defects known as VCRL syndrome (Vertebral-Cardiac-Renal-Limb syndrome). Here, we analyzed the genetic variants in NADSYN1 in an exome-sequenced cohort consisting of patients with congenital vertebral malformations (CVMs). A total number of eight variants in NADSYN1, including two truncating variants and six missense variants, were identified in nine unrelated patients. All enrolled patients presented multiple organ defects, with the involvement of either the heart, kidney, limbs, or liver, as well as intraspinal deformities. An in vitro assay using COS-7 cells demonstrated either significantly reduced protein levels or disrupted enzymatic activity of the identified variants. Our findings demonstrated that functional variants in NADSYN1 were involved in the complex genetic etiology of CVMs and provided further evidence for the causative NADSYN1 variants in congenital NAD Deficiency Disorder.

A novel compound heterozygous missense mutation in ASNS broadens the spectrum of asparagine synthetase deficiency.

BACKGROUND: Asparagine synthetase deficiency (ASNSD) is a rare pediatric congenital disorder that clinically manifests into severe progressive microcephaly, global developmental delay, spastic quadriplegia, and refractory seizures. ASNSD is caused by inheritable autosomal recessive mutations in the asparagine synthetase (ASNS) gene. METHODS: We performed whole-exome sequencing using the patient's peripheral blood, and newly discovered mutations were subsequently verified in the patient's parents via Sanger sequencing. Software-based bioinformatics analyses (protein sequence conservation analysis, prediction of protein phosphorylation sites, protein structure modeling, and protein stability prediction) were performed to investigate and deduce their downstream effects. RESULTS: In this article, we summarized all the previously reported cases of ASNSD and that of a Chinese girl who was clinically diagnosed with ASNSD, which was later confirmed via genetic testing. Whole-exome sequencing revealed two compound heterozygous missense mutations within the ASNS (c.368T > C, p.F123S and c.1649G > A, p.R550H). The origin of the two mutations was also verified in the patient's parents via Sanger sequencing. The mutation c.368T > C (p.F123S) was discovered and confirmed to be novel and previously unreported. Using software-based bioinformatics analyses, we deduced that the two mutation sites are highly conserved across a wide range of species, with the ability to alter different phosphorylation sites and destabilize the ASNS protein structure. The newly identified p.F123S mutation was predicted to be the most significantly destabilizing and detrimental mutation to the ASNS protein structure, compared to all other previously reported mutations. CONCLUSION: Evidently, the presence of these compound heterozygous mutations could lead to severe clinical phenotypes and serve as a potential indicator for considerably higher risk with less optimistic prognosis in ASNSD patients.

Role of allosteric switches and adaptor domains in long-distance cross-talk and transient tunnel formation.

Transient tunnels that assemble and disassemble to facilitate passage of unstable intermediates in enzymes containing multiple reaction centers are controlled by allosteric cues. Using the 140-kDa purine biosynthetic enzyme PurL as a model system and a combination of biochemical and x-ray crystallographic studies, we show that long-distance communication between ~25-Å distal active sites is initiated by an allosteric switch, residing in a conserved catalytic loop, adjacent to the synthetase active site. Further, combinatory experiments seeded from molecular dynamics simulations help to delineate transient states that bring out the central role of nonfunctional adaptor domains. We show that carefully orchestrated conformational changes, facilitated by interplay of dynamic interactions at the allosteric switch and adaptor-domain interface, control reactivity and concomitant formation of the ammonia tunnel. This study asserts that substrate channeling is modulated by allosteric hotspots that alter protein energy landscape, thereby allowing the protein to adopt transient conformations paramount to function.

Different ways to transport ammonia in human and Mycobacterium tuberculosis NAD+ synthetases.

NAD+ synthetase is an essential enzyme of de novo and recycling pathways of NAD+ biosynthesis in Mycobacterium tuberculosis but not in humans. This bifunctional enzyme couples the NAD+ synthetase and glutaminase activities through an ammonia tunnel but free ammonia is also a substrate. Here we show that the Homo sapiens NAD+ synthetase (hsNadE) lacks substrate specificity for glutamine over ammonia and displays a modest activation of the glutaminase domain compared to tbNadE. We report the crystal structures of hsNadE and NAD+ synthetase from M. tuberculosis (tbNadE) with synthetase intermediate analogues. Based on the observed exclusive arrangements of the domains and of the intra- or inter-subunit tunnels we propose a model for the inter-domain communication mechanism for the regulation of glutamine-dependent activity and NH3 transport. The structural and mechanistic comparison herein reported between hsNadE and tbNadE provides also a starting point for future efforts in the development of anti-TB drugs.

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan.

Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to ß-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.

Structural basis of cell wall peptidoglycan amidation by the GatD/MurT complex of Staphylococcus aureus.

The peptidoglycan of Staphylococcus aureus is highly amidated. Amidation of α-D-isoglutamic acid in position 2 of the stem peptide plays a decisive role in the polymerization of cell wall building blocks. S. aureus mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin, indicating that targeting the amidation reaction could be a useful strategy to combat this pathogen. The enzyme complex that catalyzes the formation of α-D-isoglutamine in the Lipid II stem peptide was identified recently and shown to consist of two subunits, the glutamine amidotransferase-like protein GatD and the Mur ligase homolog MurT. We have solved the crystal structure of the GatD/MurT complex at high resolution, revealing an open, boomerang-shaped conformation in which GatD is docked onto one end of MurT. Putative active site residues cluster at the interface between GatD and MurT and are contributed by both proteins, thus explaining the requirement for the assembled complex to carry out the reaction. Site-directed mutagenesis experiments confirm the validity of the observed interactions. Small-angle X-ray scattering data show that the complex has a similar conformation in solution, although some movement at domain interfaces can occur, allowing the two proteins to approach each other during catalysis. Several other Gram-positive pathogens, including Streptococcus pneumoniae, Clostridium perfringens and Mycobacterium tuberculosis have homologous enzyme complexes. Combined with established biochemical assays, the structure of the GatD/MurT complex provides a solid basis for inhibitor screening in S. aureus and other pathogens.

Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii.

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.

Determination of the formylglycinamide ribonucleotide amidotransferase ammonia pathway by combining 3D-RISM theory with experiment.

Molecular tunnels in enzyme systems possess variable architecture and are therefore difficult to predict. In this work, we design and apply an algorithm to resolve the pathway followed by ammonia using the bifunctional enzyme formylglycinamide ribonucleotide amidotransferase (FGAR-AT) as a model system. Though its crystal structure has been determined, an ammonia pathway connecting the glutaminase domain to the 30 Å distal FGAR/ATP binding site remains elusive. Crystallography suggested two purported paths: an N-terminal-adjacent path (path 1) and an auxiliary ADP-adjacent path (path 2). The algorithm presented here, RismPath, which enables fast and accurate determination of solvent distribution inside a protein channel, predicted path 2 as the preferred mode of ammonia transfer. Supporting experimental studies validate the identity of the path, and results lead to the conclusion that the residues in the middle of the channel do not partake in catalytic coupling and serve only as channel walls facilitating ammonia transfer.

Importance of hydrophobic cavities in allosteric regulation of formylglycinamide synthetase: insight from xenon trapping and statistical coupling analysis.

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Šfrom the active centers. Interestingly, correlation analysis corroborated the experimental findings, and revealed that amino acids lining the functionally important cavities form correlated sets (co-evolving residues) that connect these regions to the amidotransferase active center. It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot. In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

Substrate activation and conformational dynamics of guanosine 5'-monophosphate synthetase.

Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. Guanosine 5'-monophosphate synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of xanthosine 5'-monophosphate (XMP). In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid transfer of ammonia to this intermediate. Isolation and use of the adenylylated nucleotide intermediate allowed the study of the amido transfer reaction distinct from the ATP-dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear to be faster than steady-state turnover, implying that release is likely rate-limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.

Formylglycinamide ribonucleotide amidotransferase from Salmonella typhimurium: role of ATP complexation and the glutaminase domain in catalytic coupling.

Formylglycinamide ribonucleotide (FGAR) amidotransferase (FGAR-AT) takes part in purine biosynthesis and is a multidomain enzyme with multiple spatially separated active sites. FGAR-AT contains a glutaminase domain that is responsible for the generation of ammonia from glutamine. Ammonia is then transferred via a channel to a second active site located in the synthetase domain and utilized to convert FGAR to formylglycinamidine ribonucleotide (FGAM) in an adenosine triphosphate (ATP) dependent reaction. In some ammonia-channelling enzymes ligand binding triggers interdomain signalling between the two diverse active centres and also assists in formation of the ammonia channel. Previously, the structure of FGAR-AT from Salmonella typhimurium containing a glutamyl thioester intermediate covalently bound in the glutaminase active site was determined. In this work, the roles played by various ligands of FGAR-AT in inducing catalytic coupling are investigated. Structures of FGAR-AT from S. typhimurium were determined in two different states: the unliganded form and the binary complex with an ATP analogue in the presence of the glutamyl thioester intermediate. The structures were compared in order to decipher the roles of these two states in interdomain communication. Using a process of elimination, the results indicated that binding of FGAR is most likely to be the major mechanism by which catalytic coupling occurs. This is because conformational changes do not occur either upon formation of the glutamyl thioester intermediate or upon subsequent ATP complexation. A model of the FGAR-bound form of the enzyme suggested that the loop in the synthetase domain may be responsible for initiating catalytic coupling via its interaction with the N-terminal domain.

The arginine attenuator peptide interferes with the ribosome peptidyl transferase center.

The fungal arginine attenuator peptide (AAP) is encoded by a regulatory upstream open reading frame (uORF). The AAP acts as a nascent peptide within the ribosome tunnel to stall translation in response to arginine (Arg). The effect of AAP and Arg on ribosome peptidyl transferase center (PTC) function was analyzed in Neurospora crassa and wheat germ translation extracts using the transfer of nascent AAP to puromycin as an assay. In the presence of a high concentration of Arg, the wild-type AAP inhibited PTC function, but a mutated AAP that lacked stalling activity did not. While AAP of wild-type length was most efficient at stalling ribosomes, based on primer extension inhibition (toeprint) assays and reporter synthesis assays, a window of inhibitory function spanning four residues was observed at the AAP's C terminus. The data indicate that inhibition of PTC function by the AAP in response to Arg is the basis for the AAP's function of stalling ribosomes at the uORF termination codon. Arg could interfere with PTC function by inhibiting peptidyltransferase activity and/or by restricting PTC A-site accessibility. The mode of PTC inhibition appears unusual because neither specific amino acids nor a specific nascent peptide chain length was required for AAP to inhibit PTC function.

Arginine changes the conformation of the arginine attenuator peptide relative to the ribosome tunnel.

The fungal arginine attenuator peptide (AAP) is a regulatory peptide that controls ribosome function. As a nascent peptide within the ribosome exit tunnel, it acts to stall ribosomes in response to arginine (Arg). We used three approaches to probe the molecular basis for stalling. First, PEGylation assays revealed that the AAP did not undergo overall compaction in the tunnel in response to Arg. Second, site-specific photocross-linking showed that Arg altered the conformation of the wild-type AAP, but not of nonfunctional mutants, with respect to the tunnel. Third, using time-resolved spectral measurements with a fluorescent probe placed in the nascent AAP, we detected sequence-specific changes in the disposition of the AAP near the peptidyltransferase center in response to Arg. These data provide evidence that an Arg-induced change in AAP conformation and/or environment in the ribosome tunnel is important for stalling.

Structure of N-formylglycinamide ribonucleotide amidotransferase II (PurL) from Thermus thermophilus HB8.

The crystal structure of PurL from Thermus thermophilus HB8 (TtPurL; TTHA1519) was determined in complex with an adenine nucleotide, PO(4)(3-) and Mg(2+) at 2.35 Å resolution. TtPurL consists of 29 α-helices and 28 ß-strands, and one loop is disordered. TtPurL consists of four domains, A1, A2, B1 and B2, and the structures of the A1-B1 and A2-B2 domains were almost identical to each other. Although the sequence identity between TtPurL and PurL from Thermotoga maritima (TmPurL) is higher than that between TtPurL and the PurL domain of the large PurL from Salmonella typhimurium (StPurL), the secondary structure of TtPurL is much more similar to that of StPurL than to that of TmPurL.

Purine biosynthesis in archaea: variations on a theme.

BACKGROUND: The ability to perform de novo biosynthesis of purines is present in organisms in all three domains of life, reflecting the essentiality of these molecules to life. Although the pathway is quite similar in eukaryotes and bacteria, the archaeal pathway is more variable. A careful manual curation of genes in this pathway demonstrates the value of manual curation in archaea, even in pathways that have been well-studied in other domains. RESULTS: We searched the Integrated Microbial Genome system (IMG) for the 17 distinct genes involved in the 11 steps of de novo purine biosynthesis in 65 sequenced archaea, finding 738 predicted proteins with sequence similarity to known purine biosynthesis enzymes. Each sequence was manually inspected for the presence of active site residues and other residues known or suspected to be required for function.Many apparently purine-biosynthesizing archaea lack evidence for a single enzyme, either glycinamide ribonucleotide formyltransferase or inosine monophosphate cyclohydrolase, suggesting that there are at least two more gene variants in the purine biosynthetic pathway to discover. Variations in domain arrangement of formylglycinamidine ribonucleotide synthetase and substantial problems in aminoimidazole carboxamide ribonucleotide formyltransferase and inosine monophosphate cyclohydrolase assignments were also identified.Manual curation revealed some overly specific annotations in the IMG gene product name, with predicted proteins without essential active site residues assigned product names implying enzymatic activity (21 proteins, 2.8% of proteins inspected) or Enzyme Commission (E. C.) numbers (57 proteins, 7.7%). There were also 57 proteins (7.7%) assigned overly generic names and 78 proteins (10.6%) without E.C. numbers as part of the assigned name when a specific enzyme name and E. C. number were well-justified. CONCLUSIONS: The patchy distribution of purine biosynthetic genes in archaea is consistent with a pathway that has been shaped by horizontal gene transfer, duplication, and gene loss. Our results indicate that manual curation can improve upon automated annotation for a small number of automatically-annotated proteins and can reveal a need to identify further pathway components even in well-studied pathways.

Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide.

Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structural insight into two distinct eukaryotic stalling processes.

Crystal structure of the ATPPase subunit and its substrate-dependent association with the GATase subunit: a novel regulatory mechanism for a two-subunit-type GMP synthetase from Pyrococcus horikoshii OT3.

Guanosine 5'-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5'-monophosphate (adenyl-XMP) into guanosine 5'-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P. horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg(2+), ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.

Formylglycinamide ribonucleotide amidotransferase from Thermotoga maritima: structural insights into complex formation.

In the fourth step of the purine biosynthetic pathway, formyl glycinamide ribonucleotide (FGAR) amidotransferase, also known as PurL, catalyzes the conversion of FGAR, ATP, and glutamine to formyl glycinamidine ribonucleotide (FGAM), ADP, P i, and glutamate. Two forms of PurL have been characterized, large and small. Large PurL, present in most Gram-negative bacteria and eukaryotes, consists of a single polypeptide chain and contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with a putative ammonia channel located between the active sites of the latter two. Small PurL, present in Gram-positive bacteria and archaea, is structurally homologous to the FGAM synthetase domain of large PurL, and forms a complex with two additional gene products, PurQ and PurS. The structure of the PurS dimer is homologous with the N-terminal domain of large PurL, while PurQ, whose structure has not been reported, contains the glutaminase activity. In Bacillus subtilis, the formation of the PurLQS complex is dependent on glutamine and ADP and has been demonstrated by size-exclusion chromatography. In this work, a structure of the PurLQS complex from Thermotoga maritima is described revealing a 2:1:1 stoichiometry of PurS:Q:L, respectively. The conformational changes observed in TmPurL upon complex formation elucidate the mechanism of metabolite-mediated recruitment of PurQ and PurS. The flexibility of the PurS dimer is proposed to play a role in the activation of the complex and the formation of the ammonia channel. A potential path for the ammonia channel is identified.

Complexed structures of formylglycinamide ribonucleotide amidotransferase from Thermotoga maritima describe a novel ATP binding protein superfamily.

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. SmPurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the unliganded form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analogue, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. The availability of a ternary complex enabled mapping of the active site, thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. Surprising discoveries, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculation about the regulatory role of the auxiliary site in formation of the PurLSQ complex as well as the evolutionary relationship of PurLs from different organisms.

Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons.

For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the approximately 500-residue, approximately 50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD).