Week 5: Protein Design Pt 2

SOD1 binder peptide design

Generate binders with PepMLM

The original SOD1 sequence[1] is as follows:

>sp|P00441|SODC_HUMAN Superoxide dismutase [Cu-Zn] OS=Homo sapiens OX=9606 GN=SOD1 PE=1 SV=2
MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTS
AGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVV
HEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

The A4V mutation changes the alanine to valine at codon 4, which results in

MATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTS
AGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVV
HEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

Binder	Pseudo Perplexity
WRYPVAGLAHWE	15.095015
WRYPVAAVAHKE	11.070548
WRYPAVALRHKK	16.016262
WRYGPAALAWGE	12.227762
FLYRWLPSRRGG	-

Evaluate binders with AlphaFold3

Binder	ipTM score	Where peptide binds
WRYPVAGLAHWE	0.3	near the helix
WRYPVAAVAHKE	0.33	closer to the beta barrel
WRYPAVALRHKK	0.28	along the beta barrel
WRYGPAALAWGE	0.31	within a helix cavity
FLYRWLPSRRGG	0.29	next to the beta barrel

All of the ipTM values were higher than the known binder except for the third one, but they overall remained within a reasonable range of one another, meaning that my predicted binders may bind better than the known binder.

Evaluate properties of generated peptides in the PeptiVerse

Binder	Solubility	Hemolysis	Binding Affinity	MW	Net Charge (pH 7)
WRYPVAGLAHWE	Soluble	Non-hemolytic	Weak binding (5.888 pKd/pKi)	1484.7 Da	-0.15
WRYPVAAVAHKE	Soluble	Non-hemolytic	Weak binding (5.298 pKd/pKi)	1426.6 Da	0.85
WRYPAVALRHKK	Soluble	Non-hemolytic	Weak binding (5.522 pKd/pKi)	1524.8 Da	3.84
WRYGPAALAWGE	Soluble	Non-hemolytic	Weak binding (6.051 pKd/pKi)	1376.5 Da	-0.23
FLYRWLPSRRGG	Soluble	Non-hemolytic	Weak binding (5.968 pKd/pKi)	1507.7 Da	2.76

I was surprised to see that besides the fourth generated binder, the ipTM score had an inverse relationship with the binding affinity observed with PeptiVerse. All of my binderse were once again fairly similar. For these reasons, I chose to further explore the fourth peptide.

Generate optimized peptides with moPPIt

Using moPPIt, I generated four new binders with the following properties.

Binder	Hemolysis	Solubility	Affinity
KRDKQKKKTCYV	0.983	0.917	7.452
GGHTRTRSHTYI	0.967	0.917	6.118
KYDEKEETCKQL	0.826	0.917	6.885
KRRGRKRKKTSE	0.966	1.0	7.122

These generated proteins have a higher affinity than all of the previously generated ones.