Protein Analysis Tools 2 nd April, 2012 Ansuman Chattopadhyay, PhD, Head Molecular Biology Information Service Health Sciences Library System University of Pittsburgh

What we’ll do: Brief overview of CLC Main Workbench find genomic context of a protein sequence search for the presence of conserved domains create a multiple sequence alignment plot

What we’ll do: analyze primary structure such as, hydrophobicity, hydrophylicity, antigenicity, repeat sequence detection etc. predict secondary structure predict post translational modification such as,  Phosphorylation, glycosylation, …. search for interacting partners predict domain driven protein-protein interactions

Workshop Resources

HSLS MolBio Videos

Sequence Analysis Software Suits Wisconsin GCG VectorNTI DNA STAR-LaserGene Geneious CLC Main

Why CLC Main ? Windows Mac Linux DNA, RNA, Protein, Microarray Data Analysis Regular Update HSLS Licensed

CLC Main Access HSLS CLC Main Registration  Link: Access via Pitt - Network Connect  Instruction video:

CLC Main Workbench Overview Graphical Users Interface Protein sequences Import Sequence Navigation

CLC Main Graphical User Interface (GUI)

CLC Main

Navigate a protein sequence

CLC Main –getting started (basic navigation steps): ovideos/clc-navigation-ac0312.swfhttp://media.hsls.pitt.edu/media/molbi ovideos/clc-navigation-ac0312.swf CLC Main Workbench Walkthrough (Part1): clcmain-walkthrough-part1-ac0112.swf clcmain-walkthrough-part1-ac0112.swf CLC Main Workbench Walkthrough (Part2): clcmain-walkthrough-part2-ac0112.swf clcmain-walkthrough-part2-ac0112.swf Videos

Import a Protein Sequence

Protein Sequence Human PLCg1  Refseq no: NP_  Uniprot Accession Number: P19174  FASTA file  Raw sequence CLC features: Search, Import, Create new sequence

Import a DNA /Protein sequence into CLC Main (Part1): ovideos/clc-import-part1-ac0112.swfhttp://media.hsls.pitt.edu/media/molbi ovideos/clc-import-part1-ac0112.swf Import a DNA /Protein sequence into CLC Main (Part 2): os/clc-import-part2-ac0112.swfhttp://media.hsls.pitt.edu/media/molbiovide os/clc-import-part2-ac0112.swf Videos

CLC protein sequence

Protein sequence manipulation Create a new protein with PLCg1 SH2-SH2- SH3 domains

Sequence Alignment Pair-wise Alignment  Global  Local Multiple Sequence Alignment

Sequence Alignment

Pair-wise Sequence Alignment

Multiple Sequence Alignment

Tools: ClustalW and T-coffee

PLCg1 Orthologous sequences PLCg1:  Mouse: NP_  Rat: NP_  Cow: NP_  Dog: XP_  Zebra fish: NP_  Human: NP_  NP_067255,NP_037319,NP_776850,XP_542998,NP_919388,NP_002651

Create a multiple sequence alignment plot using CLC(part1): part1.swf Create a multiple sequence alignment plot using CLC (part2): part2.swf Create a multiple sequence alignment plot: Compare two peptide sequences.: Videos

Starting with a short peptide sequence find:  the whole protein sequence  orthologs in other species (nematode) Tool: UCSC BLAT NCBI BLAST against SwissProt

Peptide to whole protein Peptide seq: SPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPR

Place a mRNA or peptide sequence into the human genome (BLAT): Find homologous sequences: Videos

Find homologous sequence SPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPR

Sequence Manipulation & Format Conversion Sequence Manipulation Suite  Readseq  GenePept FASTA

Hands-On Retrieve amino acid sequence present between position 25 to 45 in Sequence A (MS Word Doc)  Identify the rat gene which encodes this peptide fragment and retrieve its whole protein sequence  Find the fruit fly homolog of this protein. What % identity the fruit fly protein shares with its rat homolog? Predict potential MAPK phosphorylation sites present in the fruit fly protein

Protein Domain Search: InterPro Scan InterPro is a database of protein families, domains, regions, repeats and sites in which identifiable features found in known proteins can be applied to new protein sequences. >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRG YEWDAGDVGAAPPGAAPAPGIFSSQPG HTPHPAASRDPVARTSPLQTPAAPGAAA GPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELF RD GVNWGRIVAFFEFGGVMCVESVNREMS PLVDNIALWMTEYLNRHLHTWIQDNGG WDAFVELYGPSMRPLFDFSWLSLKTLLS LALVGACITLGAYLGHK

Videos: Find protein domains, PTM, secondary str etc: t.swf t.swf Start with a protein pattern and find what proteins posses that domain: rosite.swf rosite.swf Search for protein domains,repeats and sites: o.swf o.swf

Protein Domain Search: ScanProsite >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRG YEWDAGDVGAAPPGAAPAPGIFSSQPG HTPHPAASRDPVARTSPLQTPAAPGAAA GPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELF RD GVNWGRIVAFFEFGGVMCVESVNREMS PLVDNIALWMTEYLNRHLHTWIQDNGG WDAFVELYGPSMRPLFDFSWLSLKTLLS LALVGACITLGAYLGHK

Pattern Search [AC]-x-V-x(4)-{ED}:  This pattern is translated as: [Ala or Cys]-any-Val- any-any-any-any-{any but Glu or Asp}  F-[GSTV]-P-R-L-[G>]

Pattern Search

Protein Primary Structure Analysis Tool: ExPASy from SIB  Calculated Mol Wt  Theoritical PI  Extinction coefficients  Estimated half-life Hydropathicity plot : Kyte & DoolittleKyte & Doolittle Hydrophilicity plot: Hopp T.P., Woods K.R

Antigenic Site Prediction Tool: Emboss Antigenic >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRG YEWDAGDVGAAPPGAAPAPGIFSSQPG HTPHPAASRDPVARTSPLQTPAAPGAAA GPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELF RD GVNWGRIVAFFEFGGVMCVESVNREMS PLVDNIALWMTEYLNRHLHTWIQDNGG WDAFVELYGPSMRPLFDFSWLSLKTLLS LALVGACITLGAYLGHK

EmBoss Antigenic Antigenic predicts potentially antigenic regions of a protein sequence, using the method of Kolaskar and Tongaonkar.Analysis of data from experimentally determined antigenic sites on proteins has revealed that the hydrophobic residues Cys, Leu and Val, if they occur on the surface of a protein, are more likely to be a part of antigenic sites. A semi-empirical method which makes use of physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes was developed by Kolaskar and Tongaonkar to predict antigenic determinants on proteins. Application of this method to a large number of proteins has shown that their method can predict antigenic determinants with about 75% accuracy which is better than most of the known methods. This method is based on a single parameter and thus very simple to use.

Transmembrane Region prediction

Transmembrane Site Prediction Tool: TMHMM Server >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRG YEWDAGDVGAAPPGAAPAPGIFSSQPG HTPHPAASRDPVARTSPLQTPAAPGAAA GPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELF RD GVNWGRIVAFFEFGGVMCVESVNREMS PLVDNIALWMTEYLNRHLHTWIQDNGG WDAFVELYGPSMRPLFDFSWLSLKTLLS LALVGACITLGAYLGHK

Protein Secondary Structure >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRG YEWDAGDVGAAPPGAAPAPGIFSSQPG HTPHPAASRDPVARTSPLQTPAAPGAAA GPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELF RD GVNWGRIVAFFEFGGVMCVESVNREMS PLVDNIALWMTEYLNRHLHTWIQDNGG WDAFVELYGPSMRPLFDFSWLSLKTLLS LALVGACITLGAYLGHK

Protein-Protein Interactions Prediction Tool: STRING >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRG YEWDAGDVGAAPPGAAPAPGIFSSQPG HTPHPAASRDPVARTSPLQTPAAPGAAA GPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELF RD GVNWGRIVAFFEFGGVMCVESVNREMS PLVDNIALWMTEYLNRHLHTWIQDNGG WDAFVELYGPSMRPLFDFSWLSLKTLLS LALVGACITLGAYLGHK

Hands-on Take the human BCL2 protein sequence and  Find its domain architecture  Predict the topology of its transmembrane region  Design suitable antigenic site for antibody generation  What is its calculated Mol Wt and Ext Coefficient?  Predict its secondary structure What % of this protein possesses alpha helical structure?  Predict its potential interacting partners

Hands-on Prediction of potential phosphorylation sites present in a protein sequence. Sequence: human BCL2 >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIF SSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLR QAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRD GVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWI QDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK

Phosphorylation Site Prediction: >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRG YEWDAGDVGAAPPGAAPAPGIFSSQPG HTPHPAASRDPVARTSPLQTPAAPGAAA GPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELF RD GVNWGRIVAFFEFGGVMCVESVNREMS PLVDNIALWMTEYLNRHLHTWIQDNGG WDAFVELYGPSMRPLFDFSWLSLKTLLS LALVGACITLGAYLGHK Tool: NetPhos

Phosphorylation Site Prediction: >gi| |ref|NP_ | B-cell lymphoma protein 2 alpha isoform MAHAGRTGYDNREIVMKYIHYKLSQRG YEWDAGDVGAAPPGAAPAPGIFSSQPG HTPHPAASRDPVARTSPLQTPAAPGAAA GPALSPVPPVVHLTLRQAGDDFSRRYRR DFAEMSSQLHLTPFTARGRFATVVEELF RD GVNWGRIVAFFEFGGVMCVESVNREMS PLVDNIALWMTEYLNRHLHTWIQDNGG WDAFVELYGPSMRPLFDFSWLSLKTLLS LALVGACITLGAYLGHK Tool: GPS

Thank you! Any questions? Carrie IwemaAnsuman Chattopadhyay