Today we will use bioinformatics tools to try to predict something about the function of nud-1. To do this we will use several on-line programs.
Under BIOLOGY WORKBENCH, go to protein tools and use NDJINN to locate files containing the protein sequences of nud-1 (#9081900). When using NDJINN select GBINV to only search invertebrate sequences. Import the cDNA sequence into your account.
Translate your cDNA sequence into amino acids using SIXFRAMES. Import the longest open reading frame. Use the programs AASTATS and PI to determine the predicted the number of amino acids, molecular weight, and isoelectric point of nud-1.
Secodary Structures are alpha helices and beta sheets in proteins. Programs use different algorithms to predict these structures, but often arrive at similar conclusions. Such information can be useful in predicting the approximate structure of a protein or a particular region of interest in a protein.
The program PELE on BIOLOGY WORKBENCH is very useful in that it predicts secondary structure using eight different programs and aligns their results. This allows you to rapidly compare the results from several models. Determine if nud-1 is likely alpha-helical, beta-sheet or a combination. Nud-1 is involved in nuclear migration, which requires filamentous proteins to move the nucleus. These proteins often have long stretches of alpha-helix. Comment on nud-1's ability to interact with filamentous proteins based upon its predicted secondary structure.
Protein Motifs refer to short amino acid sequences which often give the protein a specific function. Examples include glycosylation and phosphorylation sites, as well as conserved residues in enzyme active sites. The presence of specific motifs can give a researcher clues to the function of a protein, and may be valuable in designing experiments to test the function or activity of a protein. Use the programs PROSEARCH and RPSBLAST to predict protein motifs found in nud-1.
DART Domain Architecture Retrieval Tool is another program that predicts motifs. To use it, VIEW the nud-1 amino acid sequence, copy it, and paste it into the box on DART. What domains does nud-1 have? Do any other proteins have a similar domain? Is the domain in the carboxy or amino half of nud-1?
Transmembrane Domains are hydrophobic alpha helices or beta sheets which can span lipid bilayers. It takes about 20 amino acids to span a lipid bilayer in an alpha helix. Programs can detect these transmembrane domains by looking for the presence of an alpha helix 20 amino acids long which contains hydrophobic amino acids. A transmembrane domain at the amino terminus of a protein often indicates a signal peptide, which is necessary for entry into the secretory pathway.
GREASE allows you to generate Kyte-Doolittle Hydropathy Profile. This does not predict secondary structure, so it will detect both alpha helix and beta sheet transmembrane domains. Numbers grater than 0 indicate increased hydrophobicity, numbers less than 0 indicate an increase in hydrophilic amino acids.
TMAP uses a Kyte-Doolittle Hydropathy Profile to detect transmembrane spanning domains. This does not require that the domain be an alpha helix, as in TMHMM. It also provides the amino acid numbers for the transmembrane domain.
Does nud-1 have any transmembrane domains? Would you predict it to be a cytoplasmic protein, a secreted protein, or a transmembrane protein?
Wormbase is a collection of information on C. elegans genes and proteins. Examine the information on nud-1. Homologous (similar) proteins in other species are called orthologs. Homologous proteins in the same species are called paralogs. Does nud-1 have any orthologs or paralogs? If so which have the highest degree of identity (last column). What information about the function of nud-1 can be obtained from these homologs?
At the bottom of the page is a link to references on nud-1. I gave you a copy of the only publication, but there are also links to abstracts from meetings.
The information you gather can be used in your introduction or results in your lab report.
