NITROXIDE SPIN-SPIN INTERACTIONS: Applications to Protein Structure and Dynamics

Hustedt, Eric J.; Beth, Albert H.

doi:10.1146/annurev.biophys.28.1.129

Cited by 126 publications

(117 citation statements)

References 72 publications

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“…In particular, we have used the nitroxide scanning strategy in the middle section and the C-terminal region of the core complex. Because there are two copies of syntaxin 1A in the complex, single spin labeling on syntaxin 1A renders two nitroxide spin labels, and the distance between the pairs can be determined (26,27). From EPR studies we found that the middle section of the t-SNARE bundle is a coiled coil that is identical to that of the ternary complex.…”

mentioning

confidence: 93%

The Four-helix Bundle of the Neuronal Target Membrane SNARE Complex Is Neither Disordered in the Middle nor Uncoiled at the C-terminal Region

Zhang

Chen

Kweon

et al. 2002

Journal of Biological Chemistry

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Assembly of the SNARE complex is an essential step for membrane fusion and neurotransmitter release in neurons. The plasma membrane SNAREs syntaxin 1A and SNAP-25 (t-SNAREs) and the delivery-vesicle SNARE VAMP2 (or v-SNARE) contain the "SNARE regions" that essentially mediate SNARE pairing. Using site-directed spin labeling and EPR distance measurement we show that two identical copies of the SNARE region from syntaxin 1A intertwine as a coiled coil near the "ionic layer" region. The structure of the t-SNARE complex appears to be virtually identical to that of the ternary SNARE complex, except that VAMP2 is substituted to the second copy of syntaxin 1A. Furthermore, it appears that the coiled coil structure is maintained up to residue 259 of syntaxin 1A, identical to that of the ternary complex. These results are somewhat contradictory to the previous reports, suggesting that the t-SNARE complex has the disordered midsection Neurotransmitter release at synapses requires fusion of synaptic vesicles with the target presynaptic plasma membrane. Synaptic membrane fusion is directly or indirectly mediated by a set of highly conserved proteins referred to as SNARE 1 proteins (1-3). Target (t-) SNAREs syntaxin 1A and SNAP-25 are plasma membrane associate proteins. Syntaxin 1A is a transmembrane protein in which the soluble domain protrudes from the plasma membrane, whereas SNAP-25 is a soluble protein that is bound to the membrane by lipid chains. Two t-SNAREs spontaneously form the binary complex that might serve as an intermediate for the SNARE complex assembly (4). Upon docking of the vesicle to the plasma membrane vesicle (v-) SNARE VAMP2 interacts with the t-SNARE complex to form a stable SNARE complex that perhaps leads to, or catalyzes, the fusion of two membranes.The highlight of the SNARE assembly is the formation of a parallel four-helix bundle (5, 6). The SNARE regions, which are the H3 domain of syntaxin 1A, two coiled-coil domains of SNAP-25, and a VAMP2 helical domain (7-10), assemble into a 110-Å-long parallel four-stranded coiled coil (5, 6). The parallel arrangement of syntaxin 1A and VAMP2 would bring two transmembrane domains into close proximity, setting up the stage for membrane apposition (11, 12). Based on this structure, one can envision that the SNARE assembly forces the two membranes into apposition, promoting membrane fusion. The high stability of the complex perhaps implies that the energy released from the complex formation might be used to overcome the fusion energy barrier. A similar mechanism has been proposed for viral-cellular membrane fusion systems such as flu hemagglutinin and human immunodeficiency virus gp41 (13,14). It is, however, still controversial whether the SNARE complex is actively involved in fusion (1) or whether it plays a mere catalytic role (15) or a different role (16). Nevertheless, slow but measurable membrane fusion has been observed for cell-free SNARE-reconstituted vesicles (17, 18).On the other hand, the structure of the t-SNARE complex would provide valuable i...

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mentioning

confidence: 93%

The Four-helix Bundle of the Neuronal Target Membrane SNARE Complex Is Neither Disordered in the Middle nor Uncoiled at the C-terminal Region

Zhang

Chen

Kweon

et al. 2002

Journal of Biological Chemistry

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“…Exchange decays more rapidly with distance than does the dipolar interaction. Spectral simulation shows that isotropic exchange is negligible compared with dipolar interactions for unlike nitroxides with separations in the intermediate range: r 12 = 1.28À1.56 nm [13].…”

Section: Magnetic Dipole-dipole Interaction For Like and Unlike Spinsmentioning

confidence: 94%

Intermediate dipolar distances from spin labels

Marsh

2014

Journal of Magnetic Resonance

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a b s t r a c tMethods for determining inter-spin distances between nitroxide spin labels from dipolar couplings in the intermediate range (r 12 % 1.1-2 nm) by CW-EPR are addressed. For nitroxide powder patterns, the assumption of unlike spins is a better approximation than assuming strong coupling between like spins. Methods that determine the average splitting in dipolar deconvolutions yield the mean value h1=r 3 12 i, and those that determine the dipolar contribution to the spectral second moment correspondingly yield h1=r 6 12 i. To relate these ensemble averages to the mean inter-spin distance hr 12 i requires knowledge of the distribution in r 12 . Values of the inverse roots r eff ;n ¼ h1=rÀ1=n always lie below hr 12 i. Consistent comparisons of literature data on double-labelled a-helical peptides support this view. Evaluations of r eff,n for Gaussian distributions in r 12 yield calibrations to determine hr 12 i for a given distribution width, r r 12 .

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“…For example, measurement of the distance between two spin labels in proteins permits distinguish the spatial orientation of elements of defined secondary structure (Hustedt & Beth, 1999). By using site-directed spin labeling, it is possible to determine multiple distance values and thereby build tertiary and quaternary structural models as well as measure the dynamics of structural changes.…”

Section: Nmmentioning

confidence: 99%

“…New methods have also been developed for determining interprobe distances for randomly oriented spin labels. These methods are being applied to a wide range of structural problems, including peptides, soluble proteins, and membrane proteins, that are not readily characterized by other structural techniques (Hustedt & Beth, 1999). Nevertheless, a simplemeasured d1/d parameter was used rather often during these years.…”

Section: Nmmentioning

confidence: 99%

Forty Years of the d1/d Parameter

Кокорин¹

2012

Nitroxides - Theory, Experiment and Applications

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NITROXIDE SPIN-SPIN INTERACTIONS: Applications to Protein Structure and Dynamics

Cited by 126 publications

References 72 publications

The Four-helix Bundle of the Neuronal Target Membrane SNARE Complex Is Neither Disordered in the Middle nor Uncoiled at the C-terminal Region

The Four-helix Bundle of the Neuronal Target Membrane SNARE Complex Is Neither Disordered in the Middle nor Uncoiled at the C-terminal Region

Intermediate dipolar distances from spin labels

Forty Years of the d1/d Parameter

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