Detection of threonine structural changes upon formation of the M-intermediate of bacteriorhodopsin: evidence for assignment to Thr-89

Liu, Xiaomei; Lee, Min Joo; Coleman, Matthew A.; Rath, Parshuram; Nilsson, Anders; Fischer, Wolfgang B.; Bizounok, Marina; Herzfeld, Judith; Karstens, Willem F. J.; Raap, J.; Lugtenburg, J.; Rothschild, Kenneth J.

doi:10.1016/s0005-2728(98)00088-7

Cited by 9 publications

(11 citation statements)

References 42 publications

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“…In the M intermediate of BR, some intensity changes at 1130–1110 cm –1 were observed to shift with [3- 18 O]Thr-labeling, probably originating from the COH bending of Thr . However, even with isotopic labeling, it was not possible to clearly resolve the affected bands.…”

Section: Integration and Interpretationmentioning

confidence: 95%

“…In practice, all the six carbon atoms have been labeled using [ 13 C 6 ]-His, , the three nitrogen atoms labeled using [ 15 N 3 ]-His, or all carbon and nitrogen atoms have been labeled using [ 13 C 6 , 15 N 3 ]-His . In the case of Thr, [3- 18 O]Thr has been used to assign side chain νO–D and δCOH vibrations. ,, Finally, the N–H stretch from the indole group of Trp was assigned using [indole- 15 N]Trp …”

Section: Tools For Band Assignmentmentioning

confidence: 99%

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Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

2020

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Infrared difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods, it stands out by its sensitivity to the protonation state, H-bonding, and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water molecules, or cofactors. In particular, the detection of protonation and H-bonding changes in a timeresolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution, I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the preparation of suitable samples and their characterization, strategies for the perturbation of proteins, and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focuses on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities, and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and complemented by integration and interpretation of the results in the context of the studied protein, an aspect increasingly supported by spectral calculations. Selected examples from the literature, predominately but not exclusively from retinal proteins, are used to illustrate the topics covered in this review.

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Section: Integration and Interpretationmentioning

confidence: 95%

Section: Tools For Band Assignmentmentioning

confidence: 99%

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

2020

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“…For example, the L intermediate in BR gives rise to an NH stretch mode in the tryptophan indole group at 3,477 cm −1 [50]. A positive band near 3,511 cm −1 and smaller features at 3,496 cm −1 (−) were assigned in the BR→M difference spectrum to a threonine residue (OH stretch) on the basis of [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]Thr labeling, while a much smaller band appearing at 3,486 cm −1 was tentatively assigned to a tryptophan [51] (NH stretch). The appearance of bands at the same position in AR3 indicated that similar residues undergo similar structural changes in AR3.…”

Section: Structural Changes Of Weakly Hydrogen Bonded Internal Water mentioning

confidence: 99%

Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks

et al. 2011

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Archaerhodopsin-3 (AR3) is a light-driven proton pump from Halorubrum sodomense, but little is known about its photocycle. Recent interest has focused on AR3 because of its ability to serve both as a high-performance, genetically-targetable optical silencer of neuronal activity and as a membrane voltage sensor. We examined light-activated structural changes of the protein, retinal chromophore, and internal water molecules during the photocycle of AR3. Low-temperature and rapid-scan time-resolved FTIR-difference spectroscopy revealed that conformational changes during formation of the K, M, and N photocycle intermediates are similar, although not identical, to bacteriorhodopsin (BR). Positive/negative bands in the region above 3,600 cm −1 , which have previously been assigned to structural changes of weakly hydrogen bonded internal water molecules, were substantially different between AR3 and BR. This included the absence of positive bands recently associated with a chain of proton transporting water molecules in the cytoplasmic channel and a weakly hydrogen bonded water (W401), which is part of a hydrogenbonded pentagonal cluster located near the retinal Schiff base. However, many of the broad IR continuum absorption changes below 3,000 cm −1 assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR were very similar in AR3. This work and subsequent studies comparing BR and AR3 structural changes will help identify conserved elements in BR-like proton pumps as well as bioengineer AR3 to optimize neural silencing and voltage sensing.

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“…Bands assigned to particular amino acids are equally unaffected. Examples include peaks due to Asp-85 at 1761 cm ÿ1 (þ), Asp-96 at 1742 cm ÿ1 (ÿ) (Braiman et al, 1988a;Maeda et al, 1992b), Tyr-185 at 1276 cm ÿ1 (ÿ) and 1271 cm ÿ1 (þ) (Braiman et al, 1988b;Roepe et al, 1987;Rothschild et al, 1986), Thr-89 near 1125 cm ÿ1 (Liu et al, 1998); and Trp in the 740 cm ÿ1 region (Roepe et al, 1988) and at 3486 cm ÿ1 (Maeda et al, 1992a) (data not shown). The vibrational bands assigned to at least one water molecule, most likely located in the active site of BR (Brown et al, 1994), are also not altered by the SeMet substitution (data not shown).…”

Section: Ftir Difference Spectroscopymentioning

confidence: 97%

Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

Bergo

Mamaev

Olejnik

et al. 2003

Biophysical Journal

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Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR-->M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR-->M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1,284 and 900 cm(-1) due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.

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Detection of threonine structural changes upon formation of the M-intermediate of bacteriorhodopsin: evidence for assignment to Thr-89

Cited by 9 publications

References 42 publications

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks

Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

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