Synthesis and Application of Novel Bifunctional Spin Labels

Lösel, Ralf; Philipp, Reinhard; Kálai, Tamás; Hideg, Kálmán; Trommer, Wolfgang E.

doi:10.1021/bc980138v

Cited by 18 publications

(13 citation statements)

References 20 publications

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“…DTT was removed from solution by 12 hrs dialysis against three changes of 300 mL degassed Tm buffer A, at 4°C. Following dialysis Tm was incubated with 3-fold molar excess of 3,4-Bis-(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy spin label (referred to as HO-1944 in the paper) [18], [19] for 16 hrs at 4°C. Labeled Tm was dialyzed against three changes of 300 mL Tm buffer A to remove excess label.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

et al. 2011

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The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into “ghost muscle fibers”. The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.

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Section: Methodsmentioning

confidence: 99%

Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

et al. 2011

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“…1h ). BSL, like TOAC, offers rigid and specific attachment relative to the peptide backbone 26 . When attached to a helix with Cys residues engineered at i and i + 4, BSL attains a well-defined orientation with respect to the helix axis 27 , allowing for precise measurement of helix orientation in an oriented sample 28 .…”

Section: Introductionmentioning

confidence: 99%

Structural dynamics of calmodulin-ryanodine receptor interactions: electron paramagnetic resonance using stereospecific spin labels

Her

Thompson

Karim

et al. 2018

Sci Rep

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We have used electron paramagnetic resonance, with rigid and stereospecific spin labels, to resolve structural states in calmodulin (CaM), as affected by binding of Ca and a CaM-binding peptide (RyRp) derived from the ryanodine receptor (RyR), the Ca channel that triggers muscle contraction. CaM mutants containing a pair of cysteines in the N-lobe and/or C-lobe were engineered and labeled with a stereospecifically bound bifunctional spin label (BSL). RyRp was synthesized with and without TOAC (a stereospecifically attached spin-labeled amino acid) substituted for a single amino acid near the N-terminus. Intramolecular DEER distance measurements of doubly-labeled BSL-CaM revealed that CaM exists in dynamic equilibrium among multiple states, consistent with open, closed, and compact structural models. Addition of RyRp shifted the equilibrium partially toward the compact state in the absence of Ca, and completely toward the compact state in the presence of Ca, supporting a conformational selection model. Inter-protein distance measurements show that Ca stabilizes the compact state primarily by inducing ordered binding of the CaM N-lobe to RyRp, while only slightly affecting the C-lobe. The results provide insight into the structural mechanism of CaM-mediated RyR regulation, while demonstrating the power of using two types of rigidly and stereospecifically bound spin labels.

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“…The spin-labeled amino acid TOAC provides stereospecific attachment to the peptide backbone, but this probe is currently only practical for peptides on the order of 50 amino acids or less (16). For larger proteins, spin labels have been synthesized with bulky substituents to reduce mobility (17), or substitution with additional reactive moieties to confer bifunctionality (18)(19)(20). The smallest and simplest of these derivatives shares its basic structure with the widely used methanethiosulfonate spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl methanethiosulfonate spin label (MTSSL)], with a second MTS group that allows bifunctional targeting of two Cys residues (20) (Fig.…”

mentioning

confidence: 99%

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

Binder

Cornea

Thompson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1°accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2°accuracy. We used this approach to examine the crucial ADP release step in myosin's catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures.T he myosin family of molecular motors is responsible for numerous vital functions in eukaryotes, including the contraction of striated muscle. Bundled within an intricate and highly regulated myofibril lattice, muscle myosin II converts the chemical energy released by ATP binding and hydrolysis into mechanical work, executing a series of structural transitions that generate force on actin and shorten each muscle cell (1, 2). Coupling of actin binding, nucleotide hydrolysis, and lever arm movement within myosin's catalytic domain (CD) is essential for proper function of the contractile apparatus (3, 4).Myosin function requires actin, and thus an understanding of its mechanism requires analysis of both proteins in complex. However, no crystals of actin-myosin complexes have been reported, so the resolution of actin-bound myosin structures is currently limited to that of electron microscopy. Furthermore, X-ray crystallography and electron microscopy produce only static structures in frozen or crystalline environments, which cannot accurately render the dynamics, disorder, and structural transitions that are essential to understanding function and pathology (4, 5).In contrast, site-directed spectroscopy can be used to examine the actin-myosin complex under more physiological conditions. Both fluorescence and electron paramagnetic resonance (EPR) have been used in complement to examine the structural dynamics of myosin bound to actin (6, 7). EPR offers superior orientational resolution, due to the high sensitivity of the EPR spectrum to alignment of a spin label in the applied magnetic field. A wellplaced spin label can provide direct information about orientation and dynamics in the vicinity of the labeling site, a strategy that has proven powerful in the study of myosin in oriented muscle fibers (7-9). However, conventional methods for site-direc...

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Synthesis and Application of Novel Bifunctional Spin Labels

Cited by 18 publications

References 20 publications

Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

Structural dynamics of calmodulin-ryanodine receptor interactions: electron paramagnetic resonance using stereospecific spin labels

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

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