Dynamics of Protein and Chromophore Structural Changes in the Photocycle of Photoactive Yellow Protein Monitored by Time-Resolved Optical Rotatory Dispersion^,

Abstract

The dynamics of the PYP photocycle have been studied using time-resolved optical rotatory dispersion (TRORD) spectroscopy in the visible and far-UV spectral regions to probe the changes in the chromophore configuration and the protein secondary structure, respectively. The changes in the secondary structure in PYP upon photoisomerization of the chromophore can be described by two exponential lifetimes of 2 +/- 0.8 and 650 +/- 100 ms that correspond to unfolding and refolding processes, respectively. The TRORD experiments that follow the dynamics of the chromophore report three exponential components, with lifetimes of 10 +/- 3 micros, 1.5 +/- 0.5 ms, and 515 +/- 110 ms. A comparison of the kinetic behaviors of the chromophore and protein shows that during the decay of pR(465) an initial relaxation that is localized to the chromophore hydrophobic pocket precedes the formation of the chromophore and protein structures found in pB(355). In contrast, the protein and chromophore processes occur with similar time constants during inactivation of the signaling state.

The Short-Lived Signaling State of the Photoactive Yellow Protein Photoreceptor Revealed by Combined Structural Probes

“…Previous work has indicated loss of α-helical content for the N-terminal region. [17][18][19][20][21][22][23] Our results are fully compatible with this view. Furthermore, part of the N terminus must extend away from the main core, as is evident from the increased D max parameter (maximal dimension), which is also supported by the DEER results as well as the explicit structure calculations (Figures 3, 4).…”

“…Particularly, double electron electron resonance spectroscopy (DEER) is increasingly used to probe biomolecular structure. [10][11][12][13] Extensive investigations have addressed the structural aspects of the signalling mechanism of the photoactive yellow protein (PYP) photoreceptor, [1][2][3][4][14][15][16][17][18][19][20][21][22][23][24][25][26] a 14.5 kDa cytoplasmic photoreceptor, considered to be responsible for the negative phototactic response of Halorhodospira halophila. [14] The transient structural changes that occur during its photocycle have been a focus of structural biology studies.…”

“…[1,3,18] Yet, the details of these structural rearrangements for the crucial intermediate in solution, the signaling state I 2 ′ (also referred to as the 'pB' state) have remained unresolved. In solution, the I 2 ′ state has the characteristics of a partially unfolded protein, as judged from NMR observables [17,19,20] [ Figure 1A, and Figure S1 in Supporting Information (SI)], the loss of secondary structure elements observed by FTIR and CD spectroscopy, [18,21] time-resolved ORD spectroscopy [23] and altered hydrophobic dye binding and thermodynamic observables. [22,24,25] CD studies as well as stopped flow absorption spectroscopy have shown that the ground state recovery reaction matches folding reactions of the photoreceptor.…”

Predicting the reaction coordinates of millisecond light-induced conformational changes in photoactive yellow protein

“…Furthermore, the fluorescence of the single tryptophan in PYP is quenched by energy transfer to pCA and strongly reflects changes in orientation of either pCA or Trp119 (32). Other techniques, such as time-resolved THz spectroscopy (33) and time-resolved optical rotatory dispersion (34), might be sensitive enough to detect the intermediates. The prediction that a salt bridge is correlated with the unfolding route can also be addressed experimentally.…”

Co‐refolding of two peptide fragments derived from Agrobacterium tumefaciens β‐glucosidase with catalytic activity

“…Significant inhibition was observed around 1 M of NaCl (Fig. 3(c)) suggesting that two co-refolded enzymes might have weaker ionic interactions between N-and C-terminal fragments than the native enzyme [14,15].…”