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1H-n.m.r. studies of the fibronectin 13 kDa collagen-binding fragment. Evidence for autonomous conserved type I and type II domain folds.

ABSTRACT: A 1H-n.m.r. study of a 117-residue (13 kDa) gelatin-binding fragment of human fibronectin, which contains the sixth (from the N-terminus) type I domain and the first type II domain, was undertaken. The resolution of the 1H-n.m.r. spectrum indicates that the domains are independent and mobile relative to each other. Analysis of two-dimensional 1H-n.m.r. experiments recorded at 500 MHz afforded spin-system identifications for all aromatic and a number of aliphatic residues. Utilizing the fact that phenylalanine residues occur only in the type II portion of this fragment, many spin systems were localized to either the type I or the type II module via analysis of two-dimensional nuclear-Overhauser-effect (NOESY) experiments. This allowed unambiguous assignment of the two tryptophan residues, as they occur singly in each domain. Patterns of NOESY connectivities are found to be consistent with known type I and type II domain structures; this affords a number of tentative sequence-specific assignments. For both domains, evidence of conserved hydrophobic cores and secondary-structure elements is obtained. In addition, 1H-n.m.r.-monitored thermal-melting studies demonstrate conclusively that the domains are independently folded and that the type I domain has high thermal stability relative to the type II domain. This is consistent with the results of calorimetric studies, and also confirms the localization of spin systems determined from the NOESY data.

SUBMITTER: Constantine KL

PROVIDER: S-EPMC1131021 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:The zebrafish has been suggested as a model system for studying human diseases that affect nervous system function and motor output. However, few of the ion channels that control neuronal activity in zebrafish have been characterized. Here, we have identified zebrafish orthologs of voltage-dependent Kv3 (KCNC) K+ channels. Kv3 channels have specialized gating properties that facilitate high-frequency, repetitive firing in fast-spiking neurons. Mutations in human Kv3.3 cause spinocerebellar ataxia type 13 (SCA13), an autosomal dominant genetic disease that exists in distinct neurodevelopmental and neurodegenerative forms. To assess the potential usefulness of the zebrafish as a model system for SCA13, we have characterized the functional properties of zebrafish Kv3.3 channels with and without mutations analogous to those that cause SCA13. RESULTS:The zebrafish genome (release Zv8) contains six Kv3 family members including two Kv3.1 genes (kcnc1a and kcnc1b), one Kv3.2 gene (kcnc2), two Kv3.3 genes (kcnc3a and kcnc3b), and one Kv3.4 gene (kcnc4). Both Kv3.3 genes are expressed during early development. Zebrafish Kv3.3 channels exhibit strong functional and structural homology with mammalian Kv3.3 channels. Zebrafish Kv3.3 activates over a depolarized voltage range and deactivates rapidly. An amino-terminal extension mediates fast, N-type inactivation. The kcnc3a gene is alternatively spliced, generating variant carboxyl-terminal sequences. The R335H mutation in the S4 transmembrane segment, analogous to the SCA13 mutation R420H, eliminates functional expression. When co-expressed with wild type, R335H subunits suppress Kv3.3 activity by a dominant negative mechanism. The F363L mutation in the S5 transmembrane segment, analogous to the SCA13 mutation F448L, alters channel gating. F363L shifts the voltage range for activation in the hyperpolarized direction and dramatically slows deactivation. CONCLUSIONS:The functional properties of zebrafish Kv3.3 channels are consistent with a role in facilitating fast, repetitive firing of action potentials in neurons. The functional effects of SCA13 mutations are well conserved between human and zebrafish Kv3.3 channels. The high degree of homology between human and zebrafish Kv3.3 channels suggests that the zebrafish will be a useful model system for studying pathogenic mechanisms in SCA13.

Project description:Computational metabolite annotation in untargeted profiling aims at uncovering neutral molecular masses of underlying metabolites and assign those with putative identities. Existing annotation strategies rely on the observation and annotation of adducts to determine metabolite neutral masses. However, a significant fraction of features usually detected in untargeted experiments remains unannotated, which limits our ability to determine neutral molecular masses. Despite the availability of tools to annotate, relatively few of them benefit from the inherent presence of in-source fragments in liquid chromatography-electrospray ionization-mass spectrometry. In this study, we introduce a strategy to annotate in-source fragments in untargeted data using low-energy tandem mass spectrometry (MS) spectra from the METLIN library. Our algorithm, MISA (METLIN-guided in-source annotation), compares detected features against low-energy fragments from MS/MS spectra, enabling robust annotation and putative identification of metabolic features based on low-energy spectral matching. The algorithm was evaluated through an annotation analysis of a total of 140 metabolites across three different sets of biological samples analyzed with liquid chromatography-mass spectrometry. Results showed that, in cases where adducts were not formed or detected, MISA was able to uncover neutral molecular masses by in-source fragment matching. MISA was also able to provide putative metabolite identities via two annotation scores. These scores take into account the number of in-source fragments matched and the relative intensity similarity between the experimental data and the reference low-energy MS/MS spectra. Overall, results showed that in-source fragmentation is a highly frequent phenomena that should be considered for comprehensive feature annotation. Thus, combined with adduct annotation, this strategy adds a complementary annotation layer, enabling in-source fragments to be annotated and increasing putative identification confidence. The algorithm is integrated into the XCMS Online platform and is freely available at http://xcmsonline.scripps.edu .

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1H-n.m.r. studies of the fibronectin 13 kDa collagen-binding fragment. Evidence for autonomous conserved type I and type II domain folds.

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