Abstract
Because of its high pKa, arginine (Arg) is believed to be protonated even in the hydrophobic environment of the protein interior. However, our neutron crystallographic structure of photoactive yellow protein, a light sensor, demonstrated that Arg52 adopts an electrically neutral form. We also showed that the hydrogen bond between the chromophore and Glu46 is a so-called low barrier hydrogen bond (LBHB). Because both the neutral Arg and LBHB are unusual in proteins, these observations remain controversial. To validate our findings, we carried out neutron crystallographic analysis of the E46Q mutant of PYP. The resultant structure revealed that the proportion of the cationic form is higher in E46Q than in WT, although the cationic and neutral forms of Arg52 coexist in E46Q. These observations were confirmed by the occupancy of the deuterium atom bound to the Nη1 atom combined with an alternative conformation of the N(η2)D2 group comprising sp2 hybridisation. Based on these results, we propose that the formation of the LBHB decreases the proton affinity of Arg52, stabilizing the neutral form in the crystal.
Highlights
Arginines (Args) in proteins are believed to be protonated even in the hydrophobic environment of protein interiors[1], there are a few examples of electrically neutral Args in proteins[2, 3]
We carried out neutron crystallographic analysis of photoactive yellow protein (PYP) at room temperature to investigate the highly polarised hydrogen atoms involved in the hydrogen-bonding network near the chromophore
The deuterium atom is located near the centre position between Glu[46] and p-coumaric acid (pCA) in wild-type protein (WT), the deuterium atom in E46Q is covalently bound to Gln[46], with a bond length of 1.02 Å
Summary
Arginines (Args) in proteins are believed to be protonated even in the hydrophobic environment of protein interiors[1], there are a few examples of electrically neutral Args in proteins[2, 3]. Hirano and Sato investigated the migration potential of the proton between pCA and Glu[46] using the ONIOM method[16] In their calculation, the geometry of the protein moiety was fixed to that of the crystal structure; in addition, they assumed that Arg[52] was in the electrically neutral form, as reported in the neutron crystallographic analysis. The occupancies of the deuterium atom bound to the Nη1 atom combined with an alternative conformation of the N(η2)D2 group comprising sp[2] hybridisation are estimated to be 24% for WT and 67% for E46Q Based on these observations, we conclude that the hydrogen bond near the chromophore influences the pKa of Arg[52] in the crystal state of PYP
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
