The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

Rothschild, Kenneth J.

doi:10.3233/bsi-160148

Cited by 20 publications

(11 citation statements)

References 240 publications

(274 reference statements)

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“…Low-temperature spectroscopic studies have previously been extensively performed on a variety of microbial rhodopsins and provided important information. For example, the ability to trap or partially block the decay of photocycle intermediates offers a means to characterize their spectral properties for example by the application of resonance Raman, FTIR, UV–visible, and NMR. − In conjunction with the ability to crystallize some microbial rhodopsin such as BR, low temperatures provide a way to trap these proteins in pure or mixtures of different photointermediates that can be analyzed in parallel by both spectroscopy and X-ray crystallography …”

Section: Discussionmentioning

confidence: 99%

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K

Hai

Mamaeva

et al. 2017

Biochemistry

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A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel “open” state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet–visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational bands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid C=O stretching region indicate that several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 → Gln and Ser97 → Glu substitutions, residues located close to the retinylidene Schiff base, altered the K:L ratio and several of the FTIR bands in the carboxylic acid region. In the case of the Ser97 → Glu substitution, a significant red-shift of the absorption wavelength of the K and L intermediates occurs. Sequence comparisons suggest that L formation in GtACR1 at 80 K is due in part to the substitution of the highly conserved Leu or Ile at position 93 in helix 3 (BR sequence) with the homologous Met105 in GtACR1.

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Section: Discussionmentioning

confidence: 99%

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K

Hai

Mamaeva

et al. 2017

Biochemistry

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“…In the mid-twentieth century, first studies appeared that demonstrated the large potential of infrared (IR) spectroscopy for protein research [1,2]. These days, IR spectroscopy has steadily been among methods for studying protein structures at the different levels of their organization [3][4][5][6][7]. In protein studies, IR spectroscopy is predominantly used as part of absorption spectroscopy techniques (i.e., for measuring absorption spectra).…”

Section: Introductionmentioning

confidence: 99%

Measurement of the Emission Spectra of Protein Solutions in the Infrared Range. Description of the Method and Testing Using Solution of Human Interferon Gamma as an Example

Penkov

Pen’kova

2020

Front. Phys.

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This paper describes a new method for measuring the spectra of infrared radiation emitted by protein solutions in the native state without any external excitation. Radiation is detected using a vacuum Fourier-transform infrared spectrometer, and the tested sample itself is a source of radiation. The necessary conditions for detecting radiation from a sample are the use of a highly sensitive cooled detector and the presence of a cold background. In this work, the background was a black body at the boiling point of nitrogen. It is also important to select the optimal vacuum pumping depth for the spectrometer and sample thickness. Radiation occurs due to spontaneous radiative transitions from excited vibrational energy states to the ground state of molecules. The intensity of radiation is determined by the population of the respective energy states, which, according to the Boltzmann distribution, depends on temperature and frequency. Using solution of human interferon gamma as an example, it has been shown for the first time that proteins have intrinsic radiation. The described method allows detecting spectral lines with a radiation power of about 10−8 W or even less. It has also been demonstrated that emission spectroscopy offers advantages in the signal-to-noize ratio compared to absorption spectroscopy and allows analyzing the structural characteristics of a protein, in particular, providing information about its secondary structure. Another significant advantage of the method described in the article is its noninvasiveness. At the sample temperature of 25°С, emission spectra can be detected in the range from 400 to 3,600 cm−1, which covers almost the entire frequency range of existing stretching and bending vibrations of molecules. At the same time, in the fingerprint region from 500 to 1,600 cm−1 (the most informative part of the infrared spectrum), the highest sensitivity of the method is demonstrated. There is also potential for extending the available frequency range into the far infrared and terahertz ranges. Being applicable to the study of protein solutions in low concentrations, the proposed approach is not only interesting from the point of view of fundamental science but also can have applied significance in biological and medical research.

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“…Earlier studies reported the effective usage of FTIR in the characterization of recombinant protein secondary structure elements. 40 , 41 It is worth noting that the most subtle spectral region of a protein secondary structural component is the amide I band, which is usually within 1700−1600 cm −1 ; it is a result of the C = O stretch vibrations of the peptide linkages, which make up approximately 80% of a protein. The frequencies of the amide I band constituents are established to be correlated meticulously to every secondary structural element of a protein.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Expression and Biophysical Characterization of a Druggable Schistosoma mansoni Universal Stress G4LZI3 Protein

et al. 2021

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Purpose: Universal stress protein from S. mansoni, designated as G4LZI3, was previously hypothesised as a druggable target and vaccine candidate for human schistosomiasis. The purpose of this study is to characterize a purified recombinant G4LZI3 preliminarily for subsequent structural characterisation, which will provide baseline structural data for future functional studies for the discovery, design and development of new schistosomal drugs for the treatment, control and elimination of schistosomiasis. Methods: Restriction digest analysis of a GenScript-synthesised codon-optimised G4LZI3 gene construct was carried out to ascertain its integrity and size. Thereafter, the pQE30-G4LZI3 construct was transformed into an M15 bacterial expression host. Transformed cells were induced with isopropyl β-D-thiogalactoside for recombinant protein expression of an appreciable amount of pQE30-G4LZI3, which was subsequently purified with fast protein liquid chromatography and a size exclusion chromatographic purification scheme. Preliminary biophysical characterisation of the 6X His-tagged G4LZI3 was done to determine its secondary structure characteristics and protein stability. Results: A molecular weight protein of 20.3 kDa was confirmed subsequent to restriction digest analysis, while heterologous protein expression yielded a highly soluble and considerable amount of histidine-tagged G4LZI3 protein, which was successfully purified to homogeneity. Biophysical characterisation indicated that the protein was well folded, heat-stable, had the functional groups and secondary structure composition required and was thus amenable to further structural characterisation and determination. Conclusion: Biophysical characterisation of purified G4LZI3 showed that further structural studies can be embarked upon on the use of G4LZI3 as a druggable target and possibly a vaccine target against schistosomiasis via vaccinomics.

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The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

Cited by 20 publications

References 240 publications

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K

Measurement of the Emission Spectra of Protein Solutions in the Infrared Range. Description of the Method and Testing Using Solution of Human Interferon Gamma as an Example

Recombinant Expression and Biophysical Characterization of a Druggable Schistosoma mansoni Universal Stress G4LZI3 Protein

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