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A quick and simple analysis of protein secondary structure can be performed by the nnpredict tool at UCSF. (3) Notice when pasting the above sequence into the query page that there is a separate line for the name of sequence, meaning that the first line in the above fasta format sequence should be entered here, separately from the rest of the sequence. Compare the nnpredict results to the actual secondary structure of lac repressor, known from the crystal structure with PDBID 1LBI. (4)
Sequence and secondary structure - lac Repressor
Data is from PDB accession number 1LBI, RCSB Protein Data Bank.The legend for the assignments are:
H=helix; B=residue in isolated beta bridge; E=extended beta strand;G=310 helix; I=pi helix; T=hydrogen bonded turn; S=bend.
1 MKPVTLYDVA EYAGVSYQTV SRVVNQASHV SAKTREKVEA AMAELNYIPN51 RVAQQLAGKQ SLLIGVATSS LALHAPSQIV AAIKSRADQL GASVVVSMVEEEEEEEES S HHHHHHH HHHHHHHHHH T EEEEEEE
101 RSGVEACKTA VHNLLAQRVS GLIINYPLDD QDAIAVEAAC TNVPALFLDVSSHHHHHHHH HHHHHHS S EEEEES S TTHHHHHHTS SS EEESSS
151 SDQTPINSII FSHEDGTRLG VEHLVALGHQ QIALLAGPLS SVSARLRLAGTTSSS EEE E TTHHHHHH HHHHHHHT EEEEE SS SSHHHHTHHH
201 WHKYLTRNQI QPIAEREGDW SAMSGFQQTM QMLNEGIVPT AMLVANDQMAHHHHHTTTT SEEEE S SHHHHHHHHH HHHTTT S EEEESSHHHH
251 LGAMRAITES GLRVGADISV VGYDDTEDSS CYIPPLTTIK QDFRLLGQTSHHHHHHHHTT TTTBTTTEEE E SB TTGG GSSS EEE HHHHHHHH
301 VDRLLQLSQG QAVKGNQLLP VSLVKRKTTL APNTQTASPR ALADSLMQLAHHHHHHHHT S S EEE EEE TT S TTS HH HHHHHHHHHH
351 RQVSRLESGQHHHHHH
Look for regions identified as alpha-helical by nnpredict, but not identified as alpha-helical in the actual secondary structure features listed above, and vice versa. Are there any regions of 3 consecutive amino acids or more that differ? If so, how many alpha-helical regions differ and what residue numbers are involved?
Look for regions identified as beta-sheet by nnpredict, but not identified as beta-sheet in the actual secondary structure features listed above, and vice versa. Are there any regions of 3 consecutive amino acids or more that differ? If so, how many beta-sheet regions differ and what residue numbers are involved?
The PDB entry for the crystal structure of lac Repressor remarks that the N-terminal residues number 1 - 61 and the C-terminal residues number 358 - 360 are not seen in the electron density. How would this effect the assignment of actual secondary structure shown above?
A more complete sequence analysis tool that includes secondary structure prediction can be found at the PredictProtein server at Columbia University. (5) On the home page, select the tab for submission to enter a protein sequence for secondary structure prediction. The instructions indicate that you should only enter the amino acid sequence, so omit the first line when you paste in the fasta format sequence above for lac Repressor. Click on the "Results on the site, not in email" option. The server will still send an email with a link to the results. Run the prediction tool for lac Repressor sequence. When the email arrives, click on the link for your results. The secondary structure prediction algorithms are within the section entitled PROF. This is the section needed to answer the following questions.
Give a brief summary comparing the ProteinPredict results with the actual secondary structure from the PDB, listed above, and with the results from nnpredict.
With the publication of entire genomes that contain sequences to many unknown proteins, scientists would love to have the ability to predict the final folded structure of a protein based on its sequence. Although this is not yet a practical reality, tools exist that can predict secondary structure with some accuracy and inroads are being made toward solving the protein folding problem. Elucidating the mechanisms behind protein folding would provide important knowledge for fighting disease states where misfolded proteins are implicated.
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