Light-induced Conformational Changes Research Articles

Polarization-resolved surface-enhanced infrared spectra with nanosensors based on self-organized gold nanorods

Biosensors are becoming ubiquitous in the study of biomolecules, as, by modifying shape size and environment of metallic nanostructures it is now possible to engineer the field so to monitor subtle transient changes in molecular conformation at the level of a single biolayer. In this paper we present a first step towards a polarization-resolved study of light-induced conformational changes of transmembrane proteins. We exploit a platform of self-organized gold nanorods on SiO2 substrates to enhance the infrared reflection absorption spectroscopy and to perform difference spectroscopy on a light-sensitive transmembrane protein with simultaneous visible light illumination from the backside of the chip. The broad size distribution of nanorods allows us to probe with high sensitivity the modifications of the vibrational peaks over the entire fingerprint region. We show that it is possible to identify dissimilarities in the difference spectra, which in turn implies that we are monitoring over a broadband spectrum not only the chemical bonds with the dipole moment aligned orthogonally to our substrate/nanorod surface but also those with different orientation.

Interactions of drosophila cryptochrome.

In this study, we investigate the intricate regulatory mechanisms underlying the circadian clock in Drosophila, focusing on the light-induced conformational changes in the cryptochrome (DmCry). Upon light exposure, DmCry undergoes conformational changes that prompt its binding to Timeless and Jetlag proteins, initiating a cascade crucial for the starting of a new circadian cycle. DmCry is subsequently degraded, contributing to the desensitization of the resetting mechanism. The transient and short-lived nature of DmCry protein-protein interactions (PPIs), leading to DmCry degradation within an hour of light exposure, presents a challenge for comprehensive exploration. To address this, we employed proximity-dependent biotinylation techniques, combining engineered BioID (TurboID) and APEX (APEX2) enzymes with mass spectrometry. This approach enabled the identification of the invitro DmCry interactome in Drosophila S2 cells, uncovering several novel PPIs associated with DmCry. Validation of these interactions through a novel co-immunoprecipitation technique enhances the reliability of our findings. Importantly, our study suggests the potential of this method to reveal additional circadian clock- or magnetic field-dependent PPIs involving DmCry. This exploration of the DmCry interactome not only advances our understanding of circadian clock regulation but also establishes a versatile framework for future investigations into light- and time-dependent protein interactions in Drosophila.

Azobenzene as a photoswitchable mechanophore.

Azobenzene has been widely explored as a photoresponsive element in materials science. Although some studies have investigated the force-induced isomerization of azobenzene, the effect of force on the rupture of azobenzene has not been explored. Here we show that the light-induced structural change of azobenzene can also alter its rupture forces, making it an ideal light-responsive mechanophore. Using single-molecule force spectroscopy and ultrasonication, we found that cis and trans para-azobenzene isomers possess contrasting mechanical properties. Dynamic force spectroscopy experiments and quantum-chemical calculations in which azobenzene regioisomers were pulled from different directions revealed that the distinct rupture forces of the two isomers are due to the pulling direction rather than the energetic difference between the two isomers. These mechanical features of azobenzene can be used to rationally control the macroscopic fracture behaviours of polymer networks by photoillumination. The use of light-induced conformational changes to alter the mechanical response of mechanophores provides an attractive way to engineer polymer networks of light-regulatable mechanical properties.

Time-Resolved EPR Spectroscopy of Channelrhodopsin-2 Helix B Movements

The light-gated dimeric cation channel channelrhodopsin-2 (ChR2) is one of the most important optogenetic tools. Upon light activation ChR2 undergoes conformational changes, the most prominent ones include a movement of transmembrane helix B. In the present work, we apply time resolved continuous wave EPR spectroscopy to follow spectral changes of a spin label bound to position C79 located in helix B. We observed an increase of the motional freedom of the spin label side chain in illuminated ChR2. The recovery of the underlying light-induced conformational change in the dark is correlated with the recovery of the P480 state of ChR2. The observed conformational changes might be thus key elements responsible for desensitizing the channel for cation conduction.

Site-specific vibrational spectroscopy with non-canonical amino acids containing frequency-resolved labels.

Unnatural protein side-chains with frequencies within the “transparent window” spectral region (∼1800 - 2700 cm−1), where no native protein vibrations are found, can single out specific residues in vibrational spectra. These moieties contain most often triple bonds, like nitriles (R-C≡N), which can be conveniently attached to proteins in the form of genetically encoded non-canonical amino acids (ncAA) e.g. 4-cyanophenylalanine (CNF). However, the full potential of ncAA as vibrational reporters has not been exploited yet, particularly for Raman spectroscopy. Here we show how CNF residues introduced at multiple positions of the bacterial transcription factor EL222 provide a detailed picture of local changes along the photocycle. Time-resolved infrared spectra in the CNF absorption region disclose two additional kinetic events not sensed by the native probes, one occurring before flavin chromophore relaxation and the other after protein backbone relaxation. In addition, we incorporate an ncAA carrying a diacetylene (R-C≡C-C≡CH) via genetic code expansion technology into EL222 variants. The conjugated diyne features an intense Raman signal which is sensitive to the microenvironment around the triple bonds. We then measure light-induced conformational changes in the flavoprotein EL222 with single-residue precision. Our results suggest that vibrational spectroscopy assisted by ncAA can reveal protein structural dynamics residue-by-residue.

Time-resolved detection of light-induced conformational changes of heliorhodopsin.

Heliorhodopsins (HeRs) are a new category of rhodopsins. They exist as a dimer and exhibit a characteristic inverted topology. HeRs bind all-trans-retinal as a chromophore in the dark, and its isomerization to the 13-cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. The TG method reveals that the diffusion coefficient (D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. Subsequently, D decreases upon the O formation. Although two time constants (202 μs and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 μs). Light-induced unfolding of helical structure is detected by the CD method. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process.

Light-induced reversible reorganizations in closed Type II reaction centre complexes: physiological roles and physical mechanisms.

The purpose of this review is to outline our understanding of the nature, mechanism and physiological significance of light-induced reversible reorganizations in closed Type II reaction centre (RC) complexes. In the so-called 'closed' state, purple bacterial RC (bRC) and photosystem II (PSII) RC complexes are incapable of generating additional stable charge separation. Yet, upon continued excitation they display well-discernible changes in their photophysical and photochemical parameters. Substantial stabilization of their charge-separated states has been thoroughly documented-uncovering light-induced reorganizations in closed RCs and revealing their physiological importance in gradually optimizing the operation of the photosynthetic machinery during the dark-to-light transition. A range of subtle light-induced conformational changes has indeed been detected experimentally in different laboratories using different bRC and PSII-containing preparations. In general, the presently available data strongly suggest similar structural dynamics of closed bRC and PSII RC complexes, and similar physical mechanisms, in which dielectric relaxation processes and structural memory effects of proteins are proposed to play important roles.

Light-Responsive Hexagonal Assemblies of Triangular Azo Dyes.

The rational design of small building block molecules and understanding their molecular assemblies are of fundamental importance in creating new stimuli-responsive organic architectures with desired shapes and functions. Based on the experimental results of light-induced conformational changes of four types of triangular azo dyes with different terminal functional groups, as well as absorption and fluorescence characteristics associated with their molecular assemblies, we report that aggregation-active emission enhancement (AIEE)-active compound (1) substituted with sterically crowded tert-butyl (t-Bu) groups showed approximately 35% light-induced molecular switching and had a strong tendency to assemble into highly stable hexagonal structures with AIEE characteristics. Their sizes were regulated from nanometer-scale hexagonal rods to micrometer-scale sticks depending on the concentration. This is in contrast to other triangular compounds with bromo (Br) and triphenylamine (TPA) substituents, which exhibited no photoisomerization and tended to form flexible fibrous structures. Moreover, non-contact exposure of the fluorescent hexagonal nanorods to ultraviolet (UV) light led to a dramatic hexagonal-to-amorphous structure transition. The resulting remarkable variations, such as in the contrast of microscopic images and fluorescence characteristics, were confirmed by various microscopic and spectroscopic measurements.

A general approach for engineering RTKs optically controlled with far-red light

Regulation of receptor tyrosine kinase (RTK) activity is necessary for studying cell signaling pathways in health and disease. We developed a generalized approach for engineering RTKs optically controlled with far-red light. We targeted the bacterial phytochrome DrBphP to the cell surface and allowed its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. Systematic optimization of these constructs has resulted in optically regulated epidermal growth factor receptor, HER2, TrkA, TrkB, FGFR1, IR1, cKIT and cMet, named eDrRTKs. eDrRTKs induced downstream signaling in mammalian cells in tens of seconds. The ability to activate eDrRTKs with far-red light enabled spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays. We validated eDrTrkB performance in mice and found that minimally invasive stimulation in the neocortex with penetrating via skull far-red light-induced neural activity, early immediate gene expression and affected sleep patterns.

Signal transduction in light-oxygen-voltage receptors lacking the active-site glutamine

In nature as in biotechnology, light-oxygen-voltage photoreceptors perceive blue light to elicit spatiotemporally defined cellular responses. Photon absorption drives thioadduct formation between a conserved cysteine and the flavin chromophore. An equally conserved, proximal glutamine processes the resultant flavin protonation into downstream hydrogen-bond rearrangements. Here, we report that this glutamine, long deemed essential, is generally dispensable. In its absence, several light-oxygen-voltage receptors invariably retained productive, if often attenuated, signaling responses. Structures of a light-oxygen-voltage paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient light-oxygen-voltage receptors likely serve as bona fide photoreceptors, as we showcase for a diguanylate cyclase. We propose that without the glutamine, water molecules transiently approach the chromophore and thus propagate flavin protonation downstream. Signaling without glutamine appears intrinsic to light-oxygen-voltage receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.

Solid-state photoswitching in arylazopyrazole-embedded polydimethylsiloxane composite thin films

Arylazopyrazoles (AAPs) show significant potential as a new family of molecular photoswitches owing to their efficient reversible trans -to- cis photoisomerization behavior and the high thermal stability of their metastable cis -isomer. In this study, AAPs have been used for the fabrication of solid-state photoswitchable polydimethylsiloxane (PDMS) based composite thin films. The thin films were prepared by using PDMS as a polymer matrix and different concentrations of AAPs as chromophores at 150 ​°C via spin-coating. The photoswitching behavior of the AAPPDMS composite films and the pristine AAP samples induced by irradiation with specific wavelength of light were investigated. We found that the as prepared AAPPDMS composite films showed rapid and near-quantitative (>98%) reversible trans -to- cis isomerization upon alternating irradiation with UV (λ ​= ​365 ​nm) and green (λ ​= ​525 ​nm) light which is comparable to the isomerization behavior of the pristine AAP chromophores in solution. This indicates that the excellent photoswitching property of the AAPs is preserved in the solid-state of the AAP-PDMS composite films. The results also show that the optical properties of the AAP-PDMS composite films can be tuned by using different ratios of the AAP chromophores and exposure to UV-light irradiation. Additionally, the thin films were tested for their photo-actuation behavior by UV–vis spectroscopy. Irradiation of the thin film with alternating 365 ​nm UV and 525 ​nm green light lead to a slight reversible bending behavior. This is presumably caused by the light-induced conformational change of the AAP moiety embedded within the PDMS matrix and the soft nature of the PDMS. This methodology provides a new approach for exploring the fabrication of polymers with enhanced mechanical behavior and solidstate photoswitching properties. Arylazopyrazoles (AAPs) show significant potential as a new family of molecular photoswitches owing to their efficient reversible trans -tocis photoisomerization behavior and the high thermal stability of their metastable cis - isomer. In this study, AAPs have been used for the fabrication of solid-state photoswitchable polydimethylsiloxane (PDMS) based composite thin films. The thin films were prepared by using PDMS as a polymer matrix and different concentrations of AAPs as chromophores at 150 ​°C via spin-coating. The photoswitching behavior of the AAP-PDMS composite films and the pristine AAP samples induced by irradiation with specific wavelength of light were investigated. We found that the as prepared AAP-PDMS composite films showed rapid and near-quantitative (>98%) reversible trans -to- cis isomerization upon alternating irradiation with UV (λ ​= ​365 ​nm) and green (λ= 525 ​nm) light which is comparable to the isomerization behavior of the pristine AAP chromophores in solution. This indicates that the excellent photoswitching property of the AAPs is preserved in the solid-state of the AAP-PDMS composite films. The results also show that the optical properties of the AAP-PDMS composite films can be tuned by using different ratios of the AAP chromophores and exposure to UV-light irradiation. Additionally, the thin films were tested for their photo-actuation behavior by UV–vis spectroscopy. Irradiation of the thin film with alternating 365 ​nm UV and 525 ​nm green light lead to a slight reversible bending behavior. This is presumably caused by the light-induced conformational change of the AAP moiety embedded within the PDMS matrix and the soft nature of the PDMS. This methodology provides a new approach for exploring the fabrication of polymers with enhanced mechanical behavior and solid-state photoswitching properties.

Transmission of light signals from the light-oxygen-voltage core via the hydrophobic region of the \u03b2-sheet surface in aureochrome-1

Light-Oxygen-Voltage (LOV) domains are responsible for detecting blue light (BL) and regulating the activities of effector domains in various organisms. Photozipper (PZ), an N-terminally truncated aureochrome-1 protein, contains a LOV domain and a basic leucin zipper (bZIP) domain and plays a role as a light-activatable transcription factor. PZ is monomeric in the dark state and undergoes non-covalent dimerization upon illumination with BL, subsequently increasing its affinity for the target DNA. To clarify the molecular mechanism of aureochromes, we prepared site-directed mutants of PZ and performed quantitative analyses in the dark and light states. Although the amino acid substitutions in the hinge region between the LOV core and A’α helix had minor effects on the dimerization and DNA-binding properties of PZ, the substitutions in the β-sheet region of the LOV core and in the A’α helix significantly affected these properties. We found that light signals are transmitted from the LOV core to the effector bZIP domain via the hydrophobic residues on the β-sheet. The light-induced conformational change possibly deforms the hydrophobic regions of the LOV core and induces the detachment of the A’α helix to expose the dimerization surface, likely activating the bZIP domain in a light-dependent manner.

Infrared nanospectroscopy study of the light-induced conformational changes of Channelrhodopsin

The channelrhodopsin-ChR2 is a light-sensitive transmembrane protein that acts as a selective ion channel between the intra- and the extra-cellular environments. In the last decade, ChR2 has proven to be essential for optogenetics, because, if expressed in mammalian neural cells, it enables the control of neuronal activity in response to visible light. Mid-infrared difference spectroscopy can probe the functional conformational changes of light-sensitive proteins, however intrinsic limitations of standard IR spectroscopy in terms of diffraction, and therefore number of probed proteins, require that the mid-IR experiments be performed on huge numbers of lipid membrane patches with overexpressed proteins. In this work, we apply for the first time IR difference nanospectroscopy, based on the use of mid-IR lasers and an atomic force microscope (AFM), to single membrane patches containing ChR2, obtaining relevant spectroscopy results for optogenetic applications and, more generally, for future experimental studies of light-sensitive proteins at the nanoscale.

Synthetic azobenzene-containing metal-organic framework ion channels toward efficient light-gated ion transport at the subnanoscale.

Artificial nanochannels with diverse responsive properties have been widely developed to replicate the smart gating functionalities of biological ion channels. However, in these traditional nanochannels, common responsive molecules are usually too small to efficiently block the large channels under the closed states, leading to weak gating performances. Herein, we report carboxylated azobenzene-coordinated metal-organic-framework (AZO-MOF) ion channels with impressive light-gating properties. The AZO-MOF ion channels were synthesized by the confined growth of AZO-MOFs, composed of light-responsive AZO-containing ligands, non-responsive ligands and metal clusters, into ion-track-etched polymer nanochannels. The AZO-MOF ion channels with an appropriate number of AZO ligands showed a well-maintained crystalline and three-dimensional porous structure, including nanoscale cavities and subnanoscale windows for LiCl conduction. Meanwhile, the AZO-containing ligands bend and stretch upon light irradiation to open and close the pathways, thus gating the ion flux through the AZO-MOF ion channels with high on-off ratios up to 40.2, which is ∼2.3-30 times those of AZO-encapsulated MOF ion channels and AZO-modified nanochannels. This work suggests ways to achieve subnanoscaled gating of ion transport by angstrom-porous MOFs coordinated by stimuli-responsive ligands.

Photocontrolled DNA Origami Assembly by Using Two Photoswitches.

Stimuli-responsive switching molecules have been widely investigated for the purpose of the mechanical control of biomolecules. Recently developed arylazopyrazole (AAP) shows photoisomerization activity, displaying a faster response to light-induced conformational changes and unique absorption spectral properties compared with those of conventionally used azobenzene. Herein, it is demonstrated that AAP can be used as a photoswitching molecule to control photoinduced assembly and disassembly of DNA origami nanostructures. An AAP-modified DNA origami has been designed and constructed. It is observed that the repeated assembly and disassembly of AAP-modified X-shaped DNA origami and hexagonal origami with complementary strands can be achieved by alternating UV and visible-light irradiation. Closed and linear assemblies of AAP-modified X-shaped origami were successfully formed by photoirradiation, and more than 1 μm linear assemblies were formed. Finally, it is shown that the two photoswitches, AAP and azobenzene, can be used in tandem to independently control different assembly configurations by using different irradiation wavelengths. AAP can extend the variety of available wavelengths of photoswitches and stably result in the assembly and disassembly of various DNA origami nanostructures.

Tuning the redox potential of the primary electron donor in bacterial reaction centers by manganese binding and light-induced structural changes

The influence of transition metal binding on the charge storage ability of native bacterial reaction centers (BRCs) was investigated. Binding of manganous ions uniquely prevented the light-induced conformational changes that would yield to long lifetimes of the charge separated state and the drop of the redox potential of the primary electron donor (P). The lifetimes of the stable charge pair in the terminal conformations were shortened by 50-fold and 7-fold upon manganous and cupric ion binding, respectively. Nickel and zinc binding had only marginal effects. Binding of manganese not only prevented the drop of the potential of P/P+ but also elevated it by up to 117 mV depending on where the metal was binding. With variable conditions, facilitating either manganese binding or light-induced structural changes a controlled tuning of the potential of P/P+ in multiple steps was demonstrated in a range of ~200 mV without the need of a mutation or synthesis. Under the selected conditions, manganese binding was achieved without its photochemical oxidation thus, the energized but still native BRCs can be utilized in photochemistry that is not reachable with regular BRCs. A 42 Å long hydrophobic tunnel was identified that became obstructed upon manganese binding and its likely role is to deliver protons from the hydrophobic core to the surface during conformational changes.

Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality.

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ∼2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl β-d-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.

Engineering Optogenetic Protein Analogs.

This chapter provides an overview of the technologies we have developed to control proteins with light. First, we focus on the LOV domain, a versatile building block with reversible photo-response, kinetics tunable through mutagenesis, and ready expression in a broad range of cells and animals. Incorporation of LOV into proteins produced a variety of approaches: simple steric block of the active site released when irradiation lengthened a linker (PA-GTPases), reversible release from sequestration at mitochondria (LOVTRAP), and Z-lock, a method in which a light-cleavable bridge is placed where it occludes the active site. The latter two methods make use of Zdk, small engineered proteins that bind selectively to the dark state of LOV. In order to control endogenous proteins, inhibitory peptides are embedded in the LOV domain where they are exposed only upon irradiation (PKA and MLCK inhibition). Similarly, controlled exposure of a nuclear localization sequence and nuclear export sequence is used to reversibly send proteins into the nucleus. Another avenue of engineering makes use of the heterodimerization of FKBP and FRB proteins, induced by the small molecule rapamycin. We control rapamycin with light or simply add it to target cells. Incorporation of fused FKBP-FRB into kinases, guanine exchange factors, or GTPases leads to rapamycin-induced protein activation. Kinases are engineered so that they can interact with only a specific substrate upon activation. Recombination of split proteins using rapamycin-induced conformational changes minimizes spontaneous reassembly. Finally, we explore the insertion of LOV or rapamycin-responsive domains into proteins such that light-induced conformational changes exert allosteric control of the active site. We hope these design ideas will inspire new applications and broaden our reach towards dynamic biological processes that unfold when studied in vivo.

In search of visible-light photoresponsive peptide nucleic acids (PNAs) for reversible control of DNA hybridization.

Photoswitchable oligonucleotides can determine specific biological outcomes by light-induced conformational changes. In particular, artificial probes activated by visible-light irradiation are highly desired in biological applications. Here, we report two novel types of visible-light photoswitchable peptide nucleic acids (PNAs) based on the molecular transducers: hemithioindigo and tetra-ortho-fluoroazobenzene. Our study reveals that the tetra-ortho-fluoroazobenzene–PNA conjugates have promising properties (fast reversible isomerization, exceptional thermal stability, high isomer conversions and sensitivity to visible-light irradiation) as reversible modulators to control oligonucleotide hybridization in biological contexts. Furthermore, we verified that this switchable modification delivers a slightly different hybridization behavior in the PNA. Thus, both melting experiments and strand-displacement assays showed that in all the cases the trans-isomer is the one with superior binding affinities. Alternative versions, inspired by our first compounds here reported, may find applications in different fields such as chemical biology, nanotechnology and materials science.

Tip-Enhanced Infrared Difference-Nanospectroscopy of the Proton Pump Activity of Bacteriorhodopsin in Single Purple Membrane Patches.

Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using difference-spectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared difference-nanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450–1800 cm–1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.