Hoogerwerf, G. | Search bioRxiv

bioRxiv2026

Structural divergence in protein evolution of a photoreceptor family undetected by AlphaFold is observed by high sensitivity FT-IR spectroscopy

A universal mechanism in molecular evolution is functional and structural divergence of members of a protein family. The ability of AlphaFold to predict atomic-resolution protein structures promises to accelerate insights into this process. We study the interplay of changes in sequence, structure, and function in photoactive yellow protein (PYP), a family of bacterial blue light photoreceptors. Halorhodospira halophila contains two PYP homologs that diverged to 60% sequence identity, differ 100-fold in the lifetime ({tau}pB) of their pB signaling intermediate, and display altered peak wavelengths ({lambda}max) for color sensing. We resurrected ancestral PYPs and determined these properties along the resulting recapitulating evolutionary divergence. The resurrected ancestral PYP is functionally similar to PYP1, indicating divergence on the path to PYP2. AlphaFold predictions for PYP2 and these ancestral proteins revealed the absence of structural changes compared to the crystal structure of PYP1. To experimentally validate these predictions, we optimized second-derivative Fourier transform infrared (FTIR) spectroscopy. The FTIR spectra of PYP1 and 2 and their resurrected ancestral proteins demonstrated clear differences in their secondary structure. These results demonstrate an important limitation of AlphaFold and show how ancestral sequence reconstruction combined with spectroscopic approaches yields insights into divergence in a protein family.

biophysics↗

bioRxiv2025

Functional insights into the photoactive yellow protein family from homologs, multidomain proteins and inferred pyp operons

Photoactive Yellow Protein (PYP) is a model system for functional protein dynamics and a prototype of the PAS domain superfamily. It is a bacterial photoreceptor that triggers a range of responses in different bacteria: phototaxis, biosynthesis of photo-protective pigments, and light regulation of biofilm formation. An important gap in knowledge on PYP is the signal transduction chain that guides the initial signal from the photoreceptor to various biological responses. Here we report an expanded set of 984 PYP homologs, providing information on sequence conservation and variation. We analyze this set of PYPs using two bioinformatics approaches to identify candidate proteins that are functionally related to PYP. First, we identified 153 multi-domain proteins containing PYP and analyzed the domain composition of these proteins. Specific preferences for N- or C-terminal placement of the PYP domain were observed. Second, we identified 113 predicted multi-gene operons containing the pyp gene. These two approaches yielded multiple candidates for proteins in the signal transduction chain associated with PYP, particularly histidine kinase (implying phosphorylation), methyl accepting chemotaxis protein (implying phototaxis), and GGDEF and EAL proteins (implying a role of c-di-GMP and biofilm formation). Some of these candidates were present only in multi-domain proteins and others only in pyp operons. Overexpression of the PYP domain from the MCP-fusion protein from Nitrincola alkalilacustris yielded a protein with an absorbance maximum of 447 nm and an overall photocycle rate of 0.5 seconds. Our results provide a clear basis for future experimental work on identifying signal transduction partners of PYP.

microbiology↗