Length and Charge of the N-terminus Regulate the Lifetime of the Signaling State of Photoactive Yellow Protein

Abstract

Photoactive yellow protein (PYP) is one of the most extensively studied photoreceptors. Nevertheless, the role of the N-terminus in the photocycle and structural transitions is still elusive. Here, we attached additional amino acids to the N-terminus of PYP and investigated the effect of the length and charge of additional N-terminal residues using circular dichroism, two-dimensional nuclear magnetic resonance (2D-NMR), transient absorption (TA), and transient grating (TG) spectroscopic techniques. TA experiments showed that, except for negatively charged residues (5D-PYP), additional N-terminal residues of PYP generally enable faster dark recovery from the putative signaling state (pB(2)) to the ground state (pG). TG data showed that although the degree of structural changes can be controlled by adjusting specific amino acid residues in the extended N-terminus of N-terminal extended PYPs (NE-PYPs), the dark recovery times of wt-PYP and NE-PYPs, except for 5D-PYP, are independent of the structural differences between pG and pB(2) states. These results demonstrate that the recovery time and the degree of structural change can be regulated by controlling the length and sequence of N-terminal residues of PYP. The findings in this study emphasize the need for careful attention to the remaining amino acid residues when designing recombinant proteins for genetic engineering purposes.