Time-resolved Fourier Transform Infrared Spectroscopy of the Nucleotide-binding Domain from the ATP-binding Cassette Transporter MsbA

Syberg, Falk; Suveyzdis, Yan; Kötting, Carsten; Gerwert, Klaus; Hofmann, Eckhard

doi:10.1074/jbc.m112.359208

Cited by 24 publications

(27 citation statements)

References 63 publications

(56 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hence, QM/MM calculations might suggest a second γ -GTP band at 1120 cm –1 that is masked in the experiments because of the technique of difference spectroscopy. This is in agreement with two γ -phosphate bands for small GTPases and the ATPase MsbA ( 66 , 68 ). The hydrolysis spectra of G α i1 -WT actually show a band at 1120 cm −1 that was previously not assigned ( Fig.…”

Section: Discussionsupporting

confidence: 88%

See 1 more Smart Citation

Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy

Mann

Höweler

Kötting

et al. 2017

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

Time-resolved Fourier transform infrared (FTIR) spectroscopy is a powerful tool to elucidate label-free the reaction mechanisms of proteins. After assignment of the absorption bands to individual groups of the protein, the order of events during the reaction mechanism can be monitored and rate constants can be obtained. Additionally, structural information is encoded into infrared spectra and can be decoded by combining the experimental data with biomolecular simulations. We have determined recently the infrared vibrations of GTP and guanosine diphosphate (GDP) bound to Gα, a ubiquitous GTPase. These vibrations are highly sensitive for the environment of the phosphate groups and thereby for the binding mode the GTPase adopts to enable fast hydrolysis of GTP. In this study we calculated these infrared vibrations from biomolecular simulations to transfer the spectral information into a computational model that provides structural information far beyond crystal structure resolution. Conformational ensembles were generated using 15 snapshots of several 100 ns molecular-mechanics/molecular-dynamics (MM-MD) simulations, followed by quantum-mechanics/molecular-mechanics (QM/MM) minimization and normal mode analysis. In comparison with other approaches, no time-consuming QM/MM-MD simulation was necessary. We carefully benchmarked the simulation systems by deletion of single hydrogen bonds between the GTPase and GTP through several Gα point mutants. The missing hydrogen bonds lead to blue-shifts of the corresponding absorption bands. These band shifts for α-GTP (Gα-T48A), γ-GTP (Gα-R178S), and for both β-GTP/γ-GTP (Gα-K46A, Gα-D200E) were found in agreement in the experimental and the theoretical spectra. We applied our approach to open questions regarding Gα: we show that the GDP state of Gα carries a Mg, which is not found in x-ray structures. Further, the catalytic role of K46, a central residue of the P-loop, and the protonation state of the GTP are elucidated.

show abstract

Section: Discussionsupporting

confidence: 88%

“…However, the terminal P γ -O 3 group is expected to have two asymmetrical vibrations. Accordingly, in FTIR measurements of GTP in Ras ( 14 , 56 , 66 , 67 ) and ATP in MsbA ( 68 ) the γ -phosphate also showed two distinct bands in isotopic labeling experiments. The same is the case for G α i1 -GDP.…”

Section: Discussionmentioning

confidence: 94%

Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy

Mann

Höweler

Kötting

et al. 2017

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…binding of the ligand promotes the closure of the NBDs. Interestingly, in the case of the lipid exporter MsbA, the rate-limiting step corresponds to ATP cleavage, whereas for the multidrug resistance protein 4 (ABCC4), it corresponds to ADP release (47,48). However, the ATPase rates in MsbA and ABCC4 are only marginally stimulated by the substrate (47,49), similar to the mutant MalF500 in which substrate transport is largely uncoupled to ATP hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, in the case of the lipid exporter MsbA, the rate-limiting step corresponds to ATP cleavage, whereas for the multidrug resistance protein 4 (ABCC4), it corresponds to ADP release (47,48). However, the ATPase rates in MsbA and ABCC4 are only marginally stimulated by the substrate (47,49), similar to the mutant MalF500 in which substrate transport is largely uncoupled to ATP hydrolysis. Thus, although ABC transporters share similar architecture and overall mechanism, the basal ATPase activity that characterizes each transporter might determine the substrate dependence and coupling efficiency, and even perhaps the direction of transport (50).…”

Section: Discussionmentioning

confidence: 99%

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter

Bao

Dalal

Cytrynbaum

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

“…1−3 Using a pulsed light source to selectively release chemical triggers from a PPG enables potentially transformative studies of biological phenomena both in vitro and in vivo. 4 Indeed, the use of PPGs has grown rapidly, beginning with early reports describing the release of "caged" nucleotides (cAMP and ATP) from the o-nitrobenzyl chromophore 5,6 and now extending across a much broader array of PPGs that have applications in biology, 5,6 biochemistry, 7,8 and physiology. 9 The challenge of developing efficient and biologically benign PPGs is complicated by the practical requirement that the chromophore should absorb in the red or near-IR region to allow better tissue penetration and avoid unintentional photochemical reactions that occur under shorter-wavelength irradiation.…”

Section: Introductionmentioning

confidence: 99%

Two-Photon Activation of p-Hydroxyphenacyl Phototriggers: Toward Spatially Controlled Release of Diethyl Phosphate and ATP

2016

View full text Add to dashboard Cite

Two-photon activation of the p-hydroxyphenacyl (pHP) photoactivated protecting group is demonstrated for the first time using visible light at 550 nm from a pulsed laser. Broadband two-photon absorption measurements reveal a strong two-photon transition (>10 GM) near 4.5 eV that closely resembles the lowest-energy band at the same total excitation energy in the one-photon absorption spectrum of the pHP chromophore. The polarization dependence of the two-photon absorption band is consistent with excitation to the same S3 ((1)ππ*) excited state for both one- and two-photon activation. Monitoring the progress of the uncaging reaction under nonresonant excitation at 550 nm confirms a quadratic intensity dependence and that two-photon activation of the uncaging reaction is possible using visible light in the range 500-620 nm. Deprotonation of the pHP chromophore under mildly basic conditions shifts the absorption band to lower energy (3.8 eV) in both the one- and two-photon absorption spectra, suggesting that two-photon activation of the pHP chromophore may be possible using light in the range 550-720 nm. The results of these measurements open the possibility of spatially and temporally selective release of biologically active compounds from the pHP protecting group using visible light from a pulsed laser.

show abstract

Time-resolved Fourier Transform Infrared Spectroscopy of the Nucleotide-binding Domain from the ATP-binding Cassette Transporter MsbA

Cited by 24 publications

References 63 publications

Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy

Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy

Sequential Action of MalE and Maltose Allows Coupling ATP Hydrolysis to Translocation in the MalFGK2 Transporter

Two-Photon Activation of p-Hydroxyphenacyl Phototriggers: Toward Spatially Controlled Release of Diethyl Phosphate and ATP

Contact Info

Product

Resources

About