Protein Modularity and Evolution : An examination of organism complexity via protein domain structure Presented by Jennelle Heyer and Jonathan Ebbers December 7, 2004

Presentation Outline Background Material - Protein Evolution, Theory of Domains, Gene Number Hypothesis - Using a model protein family Procedure/Methods - DPIP Program, Phylogenic Analysis Results Discussion/Conclusions

Theories of Protein Evolution A long time ago, in the primodial soup of life, small polypeptides began to form … HDLC or TCP or …. HDLC + TCP = HCLCTCP HCI*CTCP + TCP … Functional proteins HDLC or TCP or …. HDLC + TCP = HCLCTCP HCI*CTCP + QZX … Functional proteins

Concept of Modularity Proteins consist of one or more domains that were pieced together over time Domain  building blocks of proteins –Defined as “ spatially distinct structures that could conceivably fold and function in isolation ” (Pontig and Russell, 2002) –Dictate the function of the protein –Evolutionary pressure to conserve (sequence and/or structure)

Organismal Complexity The nematode, C. elegans, has 19,500 genes in its genome Humans have between 20,000 and 25,000 genes in their genome HOW CAN THAT BE? Alternate splicing, multi-functional/network proteins

Hypothesis Gene products, proteins, can be multi- functional with the introduction of domains “… evolution does not produce innovation from scratch. It works on what already exists, either transforming a system to give it a new function or combining several systems to produce a more complex one ” (Jacob, 1946) More complex or phylogenetically derived organisms produce proteins with greater domain complexity

Hypothesis Part II Create a protein domain “ tool ” –Position –Partner domain –General organization –Protein evolution –Using a variety of sequenced genomes Allow investigators to learn about domain of interest and apply to research

Kinesins: A model protein family Motor proteins found in eukaryotic organisms Contain a conserved motor domain Bind and walk along microtubules Can carry a variety of “ cargo ” May contain multiple domains

Kinesins: A model protein family Arabidopsis thaliana, a model plant species, contains 61 kinesins S. pombe – 10, C. elegans – 22, Drosophlia – 25, Human and mouse ~ 45 From Reddy and Day, 2001

Programming Approach Two programs used, BLAST and InterProScan, held together with perl scripts Give a domain sequence to PSI-BLAST, which will identify proteins that have that domain. One by one, give those protein sequences to IPR, which identifies domains in the protein. Create a listing of proteins and map the data into a phylogeny. Create a tree based on the phylogeny and domains

Domain Sequence List of proteins with similar domains List of domains in every protein Tree (includes domains) BLAST InterProScan Maketree Program Flowchart

Program Details Database selection: –BLAST: Refseq over nr –InterProScan: SMART database, only Threshold values: –BLAST: Option to change, improve resolution –InterProScan: E-value at 0.99, up from 0.01 Used Arabidopsis sequences as a control Name: DPIP (Domain Placement in Proteins)

Results A Quick Look at the Data Phylogenetic Approach –Hypothesis I Qualitative Approach –Hypothesis II

A Quick L k

Phylogenetic Approach “ More complex or phylogenically derived organisms produce proteins with greater domain complexity ” Trace domain characteristics on a preset tree –Use MacClade tree drawing software –Uses input data to create most parsimonious trace Characteristics: Maximum # domains Unique domains

Maximum # of Domains per Protein Green = 1 Black = 3

Number of Unique Domains per Organism Blue = 1 Pink = 2 Dk. Blue = 3 Yellow = 5 Black = 6 Dash - ???

Phylogenetic Conclusions Inconclusive or null hypothesis supported Possible explanations: –Kinesins may have limited domain complexity due to function or folding –Inherent bias in DPIP (refseq database) Future Work: –Testing other domains through same process –Updating database –Include measure for position (N/I/C)

Qualitative Approach Create a protein domain “ tool ” –Position –Partner domain –General organization –Protein evolution –Using a variety of sequenced genomes Compile data into a more informative table

- Can I trace domain or protein evolution??

Presence of FHA/PH domain in kinesins Yellow – Absent Blue - Present

Conclusions DPIP program was created to answer two questions: –Does organismal complexity correspond with protein complexity? –Can we create a tool for researched to better understand domain in protein families? For kinesins motor domains: No and Yes For other domains:???? Thanks to Webb Miller, Richard Cyr Claude DePamphillis, Alexander Richter, Plant Physiology, Biology, and Bioinformatics Depts.