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Characterizing Changes in Peptide Binding
Specificity as S100 Proteins Evolve
1
Pollat ,
2
Wheeler ,
2
Harms
Sarina
Luke
Michael
University of New Mexico1, University of Oregon2
Specific Protein-Protein
Interactions Can Change Over Time
• Proteins can have many binding partners in a biological
environment, which may be a subset of the total possible binding
set.
• Specificity defines the interactions within the binding set.
Changing specificity can alter the binding members over
evolution.
Target 1
2. Induce bacteria
to express protein of
interest
1. Clone DNA into
vector and transform
into E. coli
3. Purify protein using
Fast Protein Liquid
Chromatography
Results and Conclusions
Kd (uM)
human
PCR product
Selected vector
Ancestor A5
Target 2
Anc A5/A6
Recombinant
vector
Protein
Plac
Target 4
Figure 1B. S100 Protein structure;
PDB 2JTT & 1K96
• We are studying how specificity has changed over the course of
evolution by using two members of the Ca2+ -binding S100
protein family, S100A5 and S100A6, as experimental models.
• Evolutionary changes in the amino acid sequence along the
S100A5 and S100A6 lineages have led to each protein
developing a different specificity for their binding partners.
tas. devil
alligator
human
tas. devil
Ancestor A6
Figure 1A. A protein’s set of binding
partners
S100A5
mouse
Target 3
Target 5
mouse
Recombinant
E. coli
•
Bacterial lysate
containing over
expressed protein
Plated on LB +
antibiotics
Purified protein
•
S100A6
alligator
chicken
The siP peptide binds to both gA6 and mA6, leading us to
believe that humans are representative of the orthologs in the
s100A6 clade. Binding of the siP peptide is conserved in
diverse species.
The NCX1 peptide binds to mA5, suggesting that binding of
this peptide is conserved in the S100A5 clade.
Kd (uM)
Ancestor A5
human
S100A5
4. We measured the binding of peptides with purified S100A5 and
S100A6 orthologous proteins using isothermal titration calorimetry
(ITC)
Future Directions
Kd (uM)
Ancestor A5/A6
human
S100A6
human
Ancestor A6
Figure 2. Human S100A5 and Human S100A6 are observed to have varying
binding specificities for two known peptides, siP and NCX1. A dark colored
diamond or rectangle is representative of strong binding, while a white color is
representative of no binding.
Ancestor A5
Anc A5/A6
• The focus of this research is to determine if the human S100A5
and human S100A6 binding patterns are representative of other
species in their clades.
Ancestor A6
mouse
?
S100A5
tas. devil ?
alligator ?
human
?
mouse
?
tas. devil ?
?
alligator ?
?
chicken
?
?
S100A6
Objectives
• To better characterize how specificity for binding partners
evolved, we will trace specificity for two known biological
targets, siP and NCX1, across orthologs and paralogs
• To do this we must obtain purified proteins from a variety of
organisms by cloning, expressing, and purifying them. We can
then do binding experiments with the purified proteins and
isolated peptides.
Figure 3. Binding of known
concentration of siP peptide to
known concentration of purified
chicken S100A6 protein.
Figure 4. Binding of known
concentration of siP peptide to
known concentration of purified
mouse S100A6 protein.
Acknowledgements
• Harms Lab: Michael Harms, Luke Wheeler, Andrea Loes, Zach Sailer, Joseph Harman, Anneliese Morrison, Bram Rickett, Ran Shi
• University Of Oregon SPUR, funded by NSF REU in Molecular Biosciences at the University of Oregon: NSF DBI/BIO 1460735
Figure 5. Binding of known
concentration of siP peptide to
known concentration of purified
chicken S100A6 protein.
• Continue peptide binding experiments for both peptides (siP,
NCX1) across all orthologs and paralogs to identify a pattern of
specificity.
• Expand the orthologous proteins (tasmanian devil, alligator) and
continue peptide binding experiments with them.
• Use other isolated peptides and test binding across all orthologs
and paralogs.