Saturation Transfer Difference Nuclear Magnetic Resonance Spectroscopy As a Method for Screening Proteins for Anesthetic Binding

Streiff, John H.; Juranić, Nenad; Macura, Slobodan; Warner, David O.; Jones, Keith A.; Perkins, William J.

doi:10.1124/mol.66.4.929

Cited by 34 publications

(38 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The STD experiments provided lines of evidence that both isoflurane and F3 directly interact with TM2e. However, the NOEs on anesthetic molecules from TM2e are small in comparison to those found from several globular proteins (39) but in a good agreement with what is observed in anesthetic interaction with transmembrane channel peptides (40). Smaller NOE on anesthetics in membrane proteins would be expected for at least two reasons.…”

Section: Discussionsupporting

confidence: 78%

NMR Study of General Anesthetic Interaction with nAChR β2 Subunit

Bondarenko

Yushmanov

et al. 2008

Biophysical Journal

View full text Add to dashboard Cite

The molecular basis of anesthetic interaction with membrane proteins has been explored via determination of anesthetic effects on the structure and dynamics of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) beta(2) subunit in dodecylphosphocholine (DPC) micelles by (1)H and (15)N solution-state NMR. Both 1-chloro-1,2,2-trifluorocyclobutane (F3) and isoflurane, two volatile general anesthetics, induced nonuniform changes in chemical shifts among residues in TM2e. Saturation transfer difference NMR experiments further confirmed the direct anesthetic interaction with TM2e. A significant and more specific anesthetic interaction was observed on three leucine residues at the helix C-terminus. Although the TM2e helical structure remained after addition of anesthetics, plausible shortening and lengthening of helix hydrogen bonds were evidenced by periodic changes in backbone amide chemical shifts. The TM2e backbone dynamics were determined on the basis of the (15)N relaxation rate constants, R(1) and R(2), and the (15)N-[(1)H] NOE using the model-free approach. The global tumbling time (11.7 ns) of TM2e in micelles slightly increased ( approximately 12.3-12.5 ns) in the presence of anesthetics. The order parameter, S(2), exceeded 0.9 for all (15)N-labeled residues, showing a restricted internal motion. Anesthetics appear to have minor effect on the TM2e's internal motion. This study provided the basis for subsequent more comprehensive studies of anesthetic effects on the transmembrane domain complex of neuronal nAChR.

show abstract

Section: Discussionsupporting

confidence: 78%

NMR Study of General Anesthetic Interaction with nAChR β2 Subunit

Bondarenko

Yushmanov

et al. 2008

Biophysical Journal

View full text Add to dashboard Cite

show abstract

“…STD has been proven to be a powerful method to probe low affinity interactions (K D ≈ 10 -8 to 10 -3 M) of small molecules with proteins (43-48). In the STD technique, selective saturation of a protein resonance leads to a rapid spread of the magnetization over the entire protein via spin diffusion, and intermolecular transfer of magnetization from protein to ligand leads to changes in NMR signal intensity of the ligand.…”

Section: Resultsmentioning

confidence: 99%

A Novel Method of Production and Biophysical Characterization of the Catalytic Domain of Yeast Oligosaccharyl Transferase

2010

View full text Add to dashboard Cite

Oligosaccharyl transferase (OT) is a multi-subunit enzyme that catalyzes N-linked glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum. In the case of Saccharomyces cerevisiae, OT is composed of nine integral membrane protein subunits. Defects in N-linked glycosylation cause a series of disorders known as congenital disorders of glycosylation (CDG). The C-terminal domain of Stt3p subunit has been reported to contain the acceptor protein recognition site and/or catalytic site. We report here the subcloning, overexpression, a robust but novel method of production of pure C-terminal domain of Stt3p at 60∼70 mg/L in E. coli. CD spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. The well dispersed 2D {1H-15N}-HSQC spectrum in SDS micelles indicates that it is feasible to determine the atomic structure by NMR. The effect of the conserved D518E mutation on the conformation of the C-terminal Stt3p is particularly interesting. The comparative analysis of the fluorescence and NMR data of the mutant and the wild-type C-terminal domain of Stt3p revealed that the replacement of the key residue Asp518, which is located within the WWDYG signature motif (residues 516-520), led to a distinct tertiary structure, even though both proteins have similar overall secondary structures. This observation strongly suggests that Asp518, which was previously proposed to primarily function as a catalytic residue, also plays a critical structural role. Moreover, the activity of the protein was confirmed by Saturation Transfer Difference (STD) and NMR titration studies.

show abstract

“…Each spectrum had 64 scans with a relaxation delay of 40 seconds. The STD (ΔI = I off − I on ) was calculated by subtraction of the intensities of on-resonance peak (I on ) from off-resonance peak (I off ) 15,16…”

Section: Methodsmentioning

confidence: 99%

Anesthetic Modulation of Protein Dynamics: Insight from an NMR Study

Canlas

Cui

et al. 2008

J. Phys. Chem. B

View full text Add to dashboard Cite

Mistic (Membrane Integrating Sequence for Translation of Integral Membrane Protein Constructs) comprises the four-α-helix bundle scaffold found in the transmembrane domains of the Cys-loop receptors that are plausible targets for general anesthetics. Nuclear magnetic resonance (NMR) studies of anesthetic halothane interaction with Mistic in dodecyl phosphocholine (DPC) micelles provide an experimental basis for understanding molecular mechanisms of general anesthesia. Halothane was found to interact directly with Mistic, mostly in the interfacial loop regions. Although the presence of halothane had little effect on Mistic structure, 15 N NMR relaxation dispersion measurements revealed that halothane affected Mistic's motion on the microsecond-millisecond timescale. Halothane shifted the equilibrium of chemical exchange in some residues and made the exchange faster or slower in comparison to the original state in the absence of halothane. The motion on the microsecond-millisecond timescale in several residues disappeared in response to the addition of halothane. Most of the residues experiencing halothane-induced dynamics changes also exhibited profound halothane-induced changes in chemical shift, suggesting that dynamics modification of these residues might result from their direct interaction with halothane molecules. Allosteric modulation by halothane also contributed to dynamics changes, as reflected in residues I52 and Y8 where halothane introduction brought about dynamics changes but not chemical shift changes. The study suggested that inhaled general anesthetics could act on proteins via altering protein motion on the microsecond-millisecond timescale, especially motion in the flexible loops that link different alpha helices. The validation of anesthetic effect on protein dynamics that are potentially correlated with protein functions is a critical step in unraveling the mechanisms of anesthetic action on proteins.

show abstract

Saturation Transfer Difference Nuclear Magnetic Resonance Spectroscopy As a Method for Screening Proteins for Anesthetic Binding

Cited by 34 publications

References 47 publications

NMR Study of General Anesthetic Interaction with nAChR β2 Subunit

NMR Study of General Anesthetic Interaction with nAChR β2 Subunit

A Novel Method of Production and Biophysical Characterization of the Catalytic Domain of Yeast Oligosaccharyl Transferase

Anesthetic Modulation of Protein Dynamics: Insight from an NMR Study

Contact Info

Product

Resources

About