Source 1primary paper2019ACS Omega
Toolkit/light-induced Fourier transform infrared (FTIR) difference spectroscopy
light-induced Fourier transform infrared (FTIR) difference spectroscopy
Also known as: FTIR difference spectroscopy
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Light-induced Fourier transform infrared (FTIR) difference spectroscopy is an assay method for detecting light-triggered structural changes associated with signaling-state formation in photoreceptor proteins. In the cited literature, it was applied to blue-light sensing LOV and BLUF/FAD systems to measure protein- and chromophore-associated spectral changes after illumination.
Usefulness & Problems
Why this is useful
This method is useful for probing photoactivated structural responses in signaling photoreceptors under illumination-dependent conditions. The supplied evidence indicates that it can resolve light-induced changes linked to flavin chemistry and helix-associated conformational responses in phototropin LOV2 and related blue-light sensors.
Source:
It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins.
Problem solved
It addresses the problem of experimentally detecting structural changes that accompany formation of the signaling state in light-responsive proteins. In the cited AsLOV2 work, it was used to assess how blue-light activation and helix architecture relate to conformational switching.
Problem links
Need conditional control of signaling activity
DerivedLight-induced Fourier transform infrared (FTIR) difference spectroscopy is an assay method for detecting light-triggered structural changes associated with signaling-state formation in photoreceptor proteins. In the cited literature, it was applied to blue-light sensing LOV and BLUF/FAD systems to measure protein- and chromophore-associated spectral changes after illumination.
Need precise spatiotemporal control with light input
DerivedLight-induced Fourier transform infrared (FTIR) difference spectroscopy is an assay method for detecting light-triggered structural changes associated with signaling-state formation in photoreceptor proteins. In the cited literature, it was applied to blue-light sensing LOV and BLUF/FAD systems to measure protein- and chromophore-associated spectral changes after illumination.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Mechanisms
allosteric conformational switchinghelix dissociation and unfoldinglight-induced difference spectroscopyphotoinduced covalent adduct formationTarget processes
signalingInput: Light
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: spectral hardware requirementoperating role: sensorswitch architecture: uncaging
The cited applications involve light-triggered measurements on flavin-binding photoreceptors, including LOV domains and a BLUF-domain protein that uses FAD. Practical details such as instrument configuration, sample preparation, illumination wavelength, and expression or delivery formats are not provided in the supplied evidence.
The supplied evidence is limited to a small number of blue-light photoreceptor contexts and does not provide general performance metrics, resolution limits, or throughput characteristics. The cited interpretation also notes that some large conformational changes, such as full Jα unfolding in truncated AsLOV2 constructs, may reflect construct artifacts rather than native full-length behavior.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Source 2primary paper2006Biochemistry
Ranked Claims
The N-terminal and C-terminal helices of phototropin LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may support signal integration of LOV1 and LOV2 domains.
It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins.
The conformational changes in full-length phototropin LOV domains may be smaller than previously assumed, and full unfolding of the Jα helix in AsLOV2 constructs with short A'α helices may be a truncation artifact.
These results are different from shorter constructs, indicating that the conformational changes in full-length phototropin LOV domains might not be as large as previously assumed, and that the well-characterized full unfolding of the Jα helix in AsLOV2 with short A'α helices may be considered a truncation artifact.
In phototropin LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces dissociation and unfolding of the C-terminal Jα helix and the N-terminal A'α helix.
In the second LOV domain of phototropins, called LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces the dissociation and unfolding of a C-terminally attached α helix (Jα) and the N-terminal helix (A'α).
Deletion of the A'α helix abolishes light-induced unfolding of the Jα helix in AsLOV2.
Deletion of the A'α helix abolishes the light-induced unfolding of Jα
Extensions of the A'α helix attenuate the light-induced structural change of the Jα helix in AsLOV2.
whereas extensions of the A'α helix lead to an attenuated structural change of Jα
The C4=O stretching bands of the FAD isoalloxazine ring were induced at the same frequency and with the same band intensity in YcgF-Full and YcgF-BLUF spectra.
the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF
The C4=O stretching bands of the FAD isoalloxazine ring were induced at the same frequency and with the same band intensity in YcgF-Full and YcgF-BLUF spectra.
the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF
The C4=O stretching bands of the FAD isoalloxazine ring were induced at the same frequency and with the same band intensity in YcgF-Full and YcgF-BLUF spectra.
the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF
The C4=O stretching bands of the FAD isoalloxazine ring were induced at the same frequency and with the same band intensity in YcgF-Full and YcgF-BLUF spectra.
the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF
The C4=O stretching bands of the FAD isoalloxazine ring were induced at the same frequency and with the same band intensity in YcgF-Full and YcgF-BLUF spectra.
the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF
The C4=O stretching bands of the FAD isoalloxazine ring were induced at the same frequency and with the same band intensity in YcgF-Full and YcgF-BLUF spectra.
the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF
The C4=O stretching bands of the FAD isoalloxazine ring were induced at the same frequency and with the same band intensity in YcgF-Full and YcgF-BLUF spectra.
the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF
Escherichia coli YcgF is a BLUF protein composed of an N-terminal BLUF domain and a C-terminal EAL domain.
The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain.
Escherichia coli YcgF is a BLUF protein composed of an N-terminal BLUF domain and a C-terminal EAL domain.
The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain.
Escherichia coli YcgF is a BLUF protein composed of an N-terminal BLUF domain and a C-terminal EAL domain.
The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain.
Escherichia coli YcgF is a BLUF protein composed of an N-terminal BLUF domain and a C-terminal EAL domain.
The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain.
Escherichia coli YcgF is a BLUF protein composed of an N-terminal BLUF domain and a C-terminal EAL domain.
The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain.
Escherichia coli YcgF is a BLUF protein composed of an N-terminal BLUF domain and a C-terminal EAL domain.
The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain.
Escherichia coli YcgF is a BLUF protein composed of an N-terminal BLUF domain and a C-terminal EAL domain.
The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity.
The light-induced FTIR difference spectrum of full-length YcgF was markedly different from that of the isolated YcgF BLUF domain, and the BLUF-domain spectrum lacked most IR bands induced in the full-length protein.
The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum.
The light-induced FTIR difference spectrum of full-length YcgF was markedly different from that of the isolated YcgF BLUF domain, and the BLUF-domain spectrum lacked most IR bands induced in the full-length protein.
The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum.
The light-induced FTIR difference spectrum of full-length YcgF was markedly different from that of the isolated YcgF BLUF domain, and the BLUF-domain spectrum lacked most IR bands induced in the full-length protein.
The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum.
The light-induced FTIR difference spectrum of full-length YcgF was markedly different from that of the isolated YcgF BLUF domain, and the BLUF-domain spectrum lacked most IR bands induced in the full-length protein.
The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum.
The light-induced FTIR difference spectrum of full-length YcgF was markedly different from that of the isolated YcgF BLUF domain, and the BLUF-domain spectrum lacked most IR bands induced in the full-length protein.
The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum.
The light-induced FTIR difference spectrum of full-length YcgF was markedly different from that of the isolated YcgF BLUF domain, and the BLUF-domain spectrum lacked most IR bands induced in the full-length protein.
The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum.
The light-induced FTIR difference spectrum of full-length YcgF was markedly different from that of the isolated YcgF BLUF domain, and the BLUF-domain spectrum lacked most IR bands induced in the full-length protein.
The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum.
YcgF-Full and YcgF-BLUF showed identical dark-state flavin UV-visible absorption spectra and identical kinetics of relaxation from the light-induced signaling state to the dark state.
YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics.
YcgF-Full and YcgF-BLUF showed identical dark-state flavin UV-visible absorption spectra and identical kinetics of relaxation from the light-induced signaling state to the dark state.
YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics.
YcgF-Full and YcgF-BLUF showed identical dark-state flavin UV-visible absorption spectra and identical kinetics of relaxation from the light-induced signaling state to the dark state.
YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics.
YcgF-Full and YcgF-BLUF showed identical dark-state flavin UV-visible absorption spectra and identical kinetics of relaxation from the light-induced signaling state to the dark state.
YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics.
YcgF-Full and YcgF-BLUF showed identical dark-state flavin UV-visible absorption spectra and identical kinetics of relaxation from the light-induced signaling state to the dark state.
YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics.
YcgF-Full and YcgF-BLUF showed identical dark-state flavin UV-visible absorption spectra and identical kinetics of relaxation from the light-induced signaling state to the dark state.
YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics.
YcgF-Full and YcgF-BLUF showed identical dark-state flavin UV-visible absorption spectra and identical kinetics of relaxation from the light-induced signaling state to the dark state.
YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics.
The full-length-specific protein bands are discussed as being predominantly attributable to structural changes in the C-terminal EAL domain triggered by light excitation of the N-terminal BLUF domain.
The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.
The full-length-specific protein bands are discussed as being predominantly attributable to structural changes in the C-terminal EAL domain triggered by light excitation of the N-terminal BLUF domain.
The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.
The full-length-specific protein bands are discussed as being predominantly attributable to structural changes in the C-terminal EAL domain triggered by light excitation of the N-terminal BLUF domain.
The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.
The full-length-specific protein bands are discussed as being predominantly attributable to structural changes in the C-terminal EAL domain triggered by light excitation of the N-terminal BLUF domain.
The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.
The full-length-specific protein bands are discussed as being predominantly attributable to structural changes in the C-terminal EAL domain triggered by light excitation of the N-terminal BLUF domain.
The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.
The full-length-specific protein bands are discussed as being predominantly attributable to structural changes in the C-terminal EAL domain triggered by light excitation of the N-terminal BLUF domain.
The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.
The full-length-specific protein bands are discussed as being predominantly attributable to structural changes in the C-terminal EAL domain triggered by light excitation of the N-terminal BLUF domain.
The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.
At medium-low temperatures, the YcgF-Full FTIR spectrum resembled the YcgF-BLUF spectrum because protein bands were selectively suppressed.
the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands
At medium-low temperatures, the YcgF-Full FTIR spectrum resembled the YcgF-BLUF spectrum because protein bands were selectively suppressed.
the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands
At medium-low temperatures, the YcgF-Full FTIR spectrum resembled the YcgF-BLUF spectrum because protein bands were selectively suppressed.
the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands
At medium-low temperatures, the YcgF-Full FTIR spectrum resembled the YcgF-BLUF spectrum because protein bands were selectively suppressed.
the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands
At medium-low temperatures, the YcgF-Full FTIR spectrum resembled the YcgF-BLUF spectrum because protein bands were selectively suppressed.
the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands
At medium-low temperatures, the YcgF-Full FTIR spectrum resembled the YcgF-BLUF spectrum because protein bands were selectively suppressed.
the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands
At medium-low temperatures, the YcgF-Full FTIR spectrum resembled the YcgF-BLUF spectrum because protein bands were selectively suppressed.
the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands
Approval Evidence
2 sources1 linked approval claimfirst-pass slugs fourier-transform-infrared-difference-spectroscopy, light-induced-fourier-transform-infrared-ftir-difference-spectroscopy
by Fourier-transform infrared difference spectroscopy
Source:
Light-induced structural changes for the signaling state formation were studied using the light-induced Fourier transform infrared (FTIR) difference spectroscopy
Source:
spectral differencesupports
The light-induced FTIR difference spectrum of full-length YcgF was markedly different from that of the isolated YcgF BLUF domain, and the BLUF-domain spectrum lacked most IR bands induced in the full-length protein.
The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum.
Source:
Comparisons
Source-backed strengths
The evidence supports sensitivity to light-induced difference signals arising from both the protein scaffold and the flavin cofactor. In Avena sativa phototropin 1 LOV2, the method supported mechanistic interpretation of blue-light-induced covalent protein-flavin bond formation together with dissociation and unfolding of the A'α and Jα helices, and it distinguished effects of A'α extension or deletion on Jα structural change.
Compared with electrical stimulation
light-induced Fourier transform infrared (FTIR) difference spectroscopy and electrical stimulation address a similar problem space because they share signaling.
Shared frame: same top-level item type; shared target processes: signaling; same primary input modality: light
Strengths here: appears more independently replicated; looks easier to implement in practice.
Compared with Förster resonance energy transfer imaging
light-induced Fourier transform infrared (FTIR) difference spectroscopy and Förster resonance energy transfer imaging address a similar problem space because they share signaling.
Shared frame: same top-level item type; shared target processes: signaling; same primary input modality: light
Strengths here: appears more independently replicated; looks easier to implement in practice.
Compared with live imaging
light-induced Fourier transform infrared (FTIR) difference spectroscopy and live imaging address a similar problem space because they share signaling.
Shared frame: same top-level item type; shared target processes: signaling; same primary input modality: light
Strengths here: appears more independently replicated; looks easier to implement in practice.
Ranked Citations
- 1.
- 2.