Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes

Abstract

Sensory rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe at the high temporal resolution inherent to IR spectroscopy.

A Critical Evaluation of Vibrational Stark Effect (VSE) Probes with the Local Vibrational Mode Theory

“…Mol. a Local mode contributions b 1-1 88.2% C1-O4, 11.8% H2-C1-H3 1-2 83.4% C1-O4, 10.4% (C1-F2, C1-F3), 6.2% F2-C1-F3 1- 3 93.1% C1-O4 1- 4 94.7% C1-O4 1- 5 85.5% C1-O4 1- 6 76.3% C3-O8, 18.7% (C3-C1, C3-C2) 1- 7 82.0% C3-O11, 6.9% C2-C3-C4 1- 8 82.4% C4-O14, 8.3% C3-C4-C5 1- 9 82.3% C1-O17, 5.2% C6-C1-C2 1- 10 77.4% C1-O2, 5.3% C3-C1-C7 1- 11 67.6% C1-O2, 7.0% C4-C1-C7, 5.6% C4-C1, 5.6% C7-C1 1- 12 77.7% C5-O10, 7.3% C4-C5-C6 1- 13 79.7% C1-O2, 6.2% C14-C1-C3 1-14 79.2% C1-O2, 6.2% C3-C1-C14 1- 15 89.2% C1-O2 1- 16 90.5% C1-O2 1- 17 71.5% C1-O16, 10.7% N11-C1-C4, 7.5% N11-C1 1- 18 80.6% C1-O2, 6.4% N3-C1, 6.4% N5-C1 1- 19 91.2% C1-O2 1-20 81.9% C5-O6, 6.1% C5-C1, 5.5% O7-C5-C1 1-21 76.0% C1-O2, 7.1% C8-C1-O3, 5.7% C8-C1 1- 22 87.2% C1-O2, 5.3% C1-C6 1- 23 75.7% C1-O2, 6.9% N7-C1, 6.8% N7-C1-C3 1- 24 93.3% C1-O2, 6.7% C1-Fe39 1- 25 94.0% C1-O2 92.5% C9-N10, 7.5% C9-S1 2-8 89.9% C1-N2, 10.1% C3-C1 2-9 45.1% N9-C7, 45.1% C8-N10 2-10 44.1% C1-N2, 44.1% C3-N4, 11.6% (C3-C7, C5-C1) 2- 11 88.3% C12-N13, 11.3% C12-C3 2- 12 88.4% N2-C1, 11.3% C3-C1 2- 13 88.3% C1-N2, 11.5% C1-C3 2-14 92.8% C13-N14, 7.2% C13-S12 2- 15 87.3% N7-C6, 12.5% C4-C6 2- 16 94.2% N2-C1, 5.8% Se3-C1 2-17 93.7% C1-N2, 6.3% C1-Se3…”

“…Below each structure, a label as n-m is given in blue, n denotes the group number (reflecting the type of VSE probe bond), and m is the number of this molecule in this group. Vibrational probe labels with superscripts are taken from the literature (a [4,10,50,51], b [10,18,24,27], c [24], d [18,50], e [5,10,18,26,27,50], f [18], g [26,50,51], h [102], i [20]). The corresponding performance score as VSE probe is given in purple.…”

“…Given a simplified electrostatic description of non-covalent interactions between the vibrational probe and surrounding molecules, the strength of these intermolecular interactions can be assessed by the electric field a target chemical bond feels, as revealed by the VSE [5,13]. The VSE has been extensively applied to study the non-covalent interactions in different types of chemical systems and environments including proteins/enzymes [6][7][8]10,11,[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33], nucleic acids [34,35], ionic liquids [36,37], biological membranes [38], electrochemical interfaces/surfaces [12,[39][40][41][42][43], and polymers [3,44,45]. Recently, the range of applications has been extended to the investigation of water clusters [46,47] and molecular solids [48].…”

Near-Infrared Activation of Sensory Rhodopsin II Mediated by NIR-to-Blue Upconversion Nanoparticles

“…Prolonged photocycle of NpSRII similar to bR is suitable for probing the photointermediate state during the photocycle with FTIR spectroscopy. Crystal structure of NpSRII (Luecke et al, 2001;Gordeliy et al, 2002) and spectroscopic studies on the photocycle of NpSRII under blue light illumination (Hirayama et al, 1992;Engelhard et al, 1996;Chizhov et al, 1998;Furutani et al, 2002;Hein et al, 2003;Furutani et al, 2004;Iwamoto et al, 2004;Bergo et al, 2005;Mironova et al, 2005;Jiang et al, 2008;Jiang et al, 2010;Tateishi et al, 2011;Mohrmann et al, 2016;Pfitzner et al, 2018) have been previously reported. Given this literature precedent, we record the FTIR difference spectrum of NpSRII under NIR light illumination.…”

Comment on “Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes”

“…In contrast to correct eq A, Mohrmann et al used an incorrect equation (eq 1 in ref 1) related to the form that describes the effect of an external electric field, F⃗ ext , on an isotropic immobilized sample. As described in detail in refs 5 and 6, complex lineshapes occur in field-on minus field-off difference spectra for an isotropic immobilized sample in an external electric field because the probe has no fixed orientation with respect to the external field; an analysis of these lineshapes gives |Δ μ⃗ probe |, the change in polarizability (typically small for vibrational transitions), and other electro-optic parameters.…”