Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion.

The heterogeneity of metabolic reactions leads to a non-uniform distribution of temperature in different parts of the living cell. The demand to study normal functioning and pathological abnormalities of cellular processes requires the development of new visualization methods. Previously, we have shown that the 35-kDa photoswitchable Orange Carotenoid Protein (OCP) has a strong temperature dependency of photoconversion rates, and its tertiary structure undergoes significant structural rearrangements upon photoactivation, which makes this protein a nano-sized temperature sensor. However, the determination of OCP conversion rates requires measurements of carotenoid absorption, which is not suitable for microscopy. In order to solve this problem, we fused green and red fluorescent proteins (TagGFP and TagRFP) to the structure of OCP, producing photoactive chimeras. In such chimeras, electronic excitation of the fluorescent protein is effectively quenched by the carotenoid in OCP. Photoactivation of OCP-based chimeras triggers rearrangements of complex geometry, permitting measurements of the conversion rates by monitoring changes of fluorescence intensity. This approach allowed us to determine the local temperature of the microenvironment. Future directions to improve the OCP-based sensor are discussed.

Comparative study of spectroscopic features reveals the structural determinants of keto-carotenoids in photoactive water-soluble proteins.

The molecular chaperone GroEL is designed to promote protein folding and prevent aggregation. However, the interaction between GroEL and the prion protein, PrP^C, could lead to pathogenic transformation of the latter to the aggregation-prone PrP^Sc form. Here, the molecular basis of the interactions in the GroEL–PrP complex is studied with cryo-EM and molecular dynamics approaches. The obtained cryo-EM structure shows PrP to be bound to several subunits of GroEL at the level of their apical domains. According to MD simulations, the disordered N-domain of PrP forms much more intermolecular contacts with GroEL. Upon binding to the GroEL, the N-domain of PrP begins to form short helices, while the C-domain of PrP exhibits a tendency to unfold its α2-helix. In the absence of the nucleotides in the system, these processes are manifested at the hundred nanoseconds to microsecond timescale.