Structural insights into the fusion of annexin A5 and fluorescent proteins generating hundredfold differentiated binding affinities to phosphatidylserine.

Fluorescent proteins (FPs) are an indispensable part of modern biology. Numerous studies utilize FPs for protein labeling and cell tracking purposes. They are commonly fused with proteins to aid in their visualization. It is generally assumed that these FP tags have minimal impact on the properties of the fusion proteins. Do the FP types affect the function and characteristics of target proteins on earth? So far, there is no definite answer. Fluorescent annexin A5 (AnxA5) has been extensively employed as apoptosis probes. However, except for chemically labeled AnxA5, there are few developed FP-based AnxA5 probes. Therefore, it is essential to screen out suitable FPs for developing high-affinity AnxA5 probes. Here, various fusion proteins (AnxA5-FPs) were developed. The fusion of AnxA5 did not change the chromophore environments of FPs, while the fusion of FPs led to over a 100-fold difference in AnxA5's affinity for phosphatidylserine (PS). We found that polymeric AnxA5-FPs had higher PS-affinity. Remarkably, although the structures of FPs were similar, they fused with AnxA5 in different modes, generating fusion proteins with different spatial conformations. The difference in conformation resulted in variations in the PS-binding pattern of AnxA5, leading to differing levels of PS-affinity. More importantly, we found five high-affinity (Kd > 10