Protein Modeling on the Internet A group of patients with Crohn disease (an infl

Question

Protein Modeling on the Internet A group of patients with Crohn disease (an inflammatory bowel disease) underwent biopsies of their intestinal mucosa in an attempt to identify the causative agent. Researchers identified a protein that was present at higher levels in patients with Crohn disease than in patients with an unrelated inflammatory bowel disease or in unaffected controls. The protein was isolated, and the following partial amino acid sequence was obtained (reads left to right):

EAELCPDRCI HSFQNLGIQC VKKRDLEQAI

SQRIQTNNNP FQVPIEEQRG DYDLNAVRLC

FQVTVRDPSG RPLRLPPVLP HPIFDNRAPN

TAELKICRVN RNSGSCLGGD EIFLLCDKVQ

KEDIEVYFTG PGWEARGSFS QADVHRQVAI

VFRTPPYADP SLQAPVRVSM QLRRPSDREL

SEPMEFQYLP DTDDRHRIEE KRKRTYETFK

SIMKKSPFSG PTDPRPPPRR IAVPSRSSAS

VPKPAPQPYP

(a) You can identify this protein using a protein database on the Internet. Some good places to start include Protein Information Resource (PIR; http://pir.georgetown.edu), Structural Classification of Proteins (SCOP; http://scop.mrc-lmb.cam.ac.uk/scop), and Prosite (http://prosite.expasy.org). At your selected database site, follow links to the sequence comparison engine. Enter about 30 residues from the protein sequence in the appropriate search field and submit it for analysis. What does this analysis tell you about the identity of the protein?

(b) Try using different portions of the amino acid sequence. Do you always get the same result?

(c) A variety of websites provide information about the three-dimensional structure of proteins. Find information about the protein’s secondary, tertiary, and quaternary structure using database sites such as the Protein Data Bank (PDB;www.pdb.org) or SCOP.

(d) In the course of your Web searches, what did you learn about the cellular function of the protein?

Explanation / Answer

(a)At the PIR International Protein Sequence Database (http://pir.georgetown.edu), click on“Search/Analysis” and choose “BLAST search.” Paste the first 30 amino acid residues ofthe sequence into the search box and submit the sequence for comparison. The table thatreturns includes many proteins that have 100% sequence identity with these 30 residues.Among the human proteins are RelA and the transcription factor NF±B. Proteins fromother species match as well. Click on the “Help” button for explanations of the variousoptions and table items.

(b)As more proteins are sequenced, the number of hits returned from a 30-residue se-quence increases. Sequence matching based on the first 30 residues brings up severalproteins that contain this sequence (identity±100%). Using sequence segments fromdifferent parts of the protein will return some different results, but the proteins withhigh sequence identities will likely be similar. Even when the entiresequence is entered in the search field, similar proteins from cattle, mouse, and rat match with very highscores, and several hundred hits are returned. When the entire sequence is used, the human protein with the best match is the p65 subunit of nuclear transcription factorkappa B (NF±B). A synonym for this protein is RelA transforming protein.

(c)At the PDB (rcsb.org) search on “NF-kappa-B p65.” You will get more than a dozen hits.Adding “human” to the search limits the results further. Go back to the more generalsearch on “NF-kappa-B p65” and scan through the returned items. NF±B has two sub-units. There are multiple variants of the subunits, with the best-characterized being 50,52, and 65 kDa (p50, p52, and p65, respectively). These pair with each other to form avariety of homodimers and heterodimers.

(d)The various proteins that predominate in this search are eukaryotic transcription factors,which stimulate transcription of genes involved in development and some immune re-sponses. The proteins have two distinct domains, including an amino-terminal Rel ho types pf proteins.their function is to stimulate transcription.

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