1. Interrogating the Dimerization Interface of the Prion Protein Via Site-Specific Mutations to p-Benzoyl-L-Phenylalanine
Sudheer Babu Sangeetham, Krisztina Huszár, Petra Bencsura, Antal Nyeste, Éva Hunyadi-Gulyás, Elfrieda Fodor, Ervin Welker J Mol Biol. 2018 Aug 17;430(17):2784-2801. doi: 10.1016/j.jmb.2018.05.027. Epub 2018 May 17.
Transmissible spongiform encephalopathies are centered on the conformational transition of the prion protein from a mainly helical, monomeric structure to a β-sheet rich ordered aggregate. Experiments indicate that the main infectious and toxic species in this process are however shorter oligomers, formation of which from the monomers is yet enigmatic. Here, we created 25 variants of the mouse prion protein site-specifically containing one genetically-incorporated para-benzoyl-phenylalanine (pBpa), a cross-linkable non-natural amino acid, in order to interrogate the interface of a prion protein-dimer, which might lie on the pathway of oligomerization. Our results reveal that the N-terminal part of the prion protein, especially regions around position 127 and 107, is integral part of the dimer interface. These together with additional pBpa-containing variants of mPrP might also facilitate to gain more structural insights into oligomeric and fibrillar prion protein species including the pathological variants.
2. Electron-deficient p-benzoyl-l-phenylalanine derivatives increase covalent chemical capture yields for protein-protein interactions
Cassandra M Joiner, Meghan E Breen, Anna K Mapp Protein Sci. 2019 Jun;28(6):1163-1170. doi: 10.1002/pro.3621. Epub 2019 Apr 29.
The photoactivatable amino acid p-benzoyl-l-phenylalanine (pBpa) has been used for the covalent capture of protein-protein interactions (PPIs) in vitro and in living cells. However, this technique often suffers from poor photocrosslinking yields due to the low reactivity of the active species. Here we demonstrate that the incorporation of halogenated pBpa analogs into proteins leads to increased crosslinking yields for protein-protein interactions. The analogs can be incorporated into live yeast and upon irradiation capture endogenous PPIs. Halogenated pBpas will extend the scope of PPIs that can be captured and expand the toolbox for mapping PPIs in their native environment.
3. p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide
J C Kauer, S Erickson-Viitanen, H R Wolfe Jr, W F DeGrado J Biol Chem. 1986 Aug 15;261(23):10695-700.
A new photoreactive amino acid analog, p-benzoyl-L-phenylalanine, is described. Convenient methods for the preparation of this amino acid and its subsequent incorporation into synthetic peptides by the solid-phase technique are outlined. To illustrate its utility, p-benzoyl-L-phenylalanine was substituted in place of tryptophan in a 17-residue calmodulin-binding peptide. The substitution did not measurably affect the affinity of this peptide for calmodulin. When this peptide was photolyzed at 350 nm in a 1:1 molar ratio with calmodulin in the presence of 500 microM CaCl2, 70% of the calmodulin was derivatized. The specificity of the reaction was investigated by photolysis in the absence of CaCl2 where little binding occurs; under these conditions little or no photolabeling occurred.
