Light scattering and the absolute characterization of macromolecules

Wyatt, Philip J.

doi:10.1016/0003-2670(93)80373-s

Cited by 1,347 publications

(1,039 citation statements)

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“…To determine the absolute molecular mass, we used MALLS, a technique that is independent of the shape of molecules (21). Synaptobrevin eluted as a monomeric species (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation

Fasshauer

Otto

Eliason

et al. 1997

Journal of Biological Chemistry

342

372

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SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors ␣-, ␤-, and ␥-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant ␣-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in ␣-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.Neurons release their neurotransmitters by the Ca 2ϩ -dependent exocytosis of synaptic vesicles. In recent years, several membrane proteins have been identified which are required for exocytotic membrane fusion. These proteins include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) 1 and the synaptic membrane proteins syntaxin and SNAP-25, collectively referred to as SNAREs. Synaptobrevin and syntaxin both contain a single transmembrane domain at the C terminus (1, 2). SNAP-25 does not contain a transmembrane domain but carries palmitoyl side chains attached to cysteine residues in the middle of the sequence (3, 4). Homologues of these proteins have been identified in many eukaryotic cells including yeast, suggesting that fusion of trafficking vesicles with their respective target membranes is mediated by a conserved mechanism (2,(5)(6)(7)(8).While the evidence linking synaptobrevin, SNAP-25, and syntaxin to exocytosis is compelling, their precise role is unknown. In detergent extracts of brain membranes, the three proteins form a tight complex (9, 10). A ternary complex with properties similar to the native complex can be formed using recombinant proteins lacking their transmembrane anchors (11). Both native and recombinant complexes can be disassembled by the concerted action of the ATPase NSF and the protein ␣-SNAP (9, 10, 12, 13). The latter two proteins are soluble, abundant, and highly conserved through evolution. They are essential for the fusion of trafficking vesicles with their target membranes (9, 10).The assembly and disassembly of the SNARE proteins has not yet been integrated into a coherent picture of exocytosis. However, any interference with these reactions seriously inhibits membrane fusion (6 -8). To further understand these essential reac...

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“…To determine the absolute molecular mass, we used MALLS, a technique that is independent of the shape of molecules (21). Synaptobrevin eluted as a monomeric species (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Light scattering and differential refractometry were carried out using the Mini-Dawn and Optilab instruments of Wyatt Technology Corp. Analysis was carried out as described by Astra software (21). For each sample, 100 l of protein solution was loaded.…”

Section: Methodsmentioning

confidence: 99%

Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation

Fasshauer

Otto

Eliason

et al. 1997

Journal of Biological Chemistry

342

372

View full text Add to dashboard Cite

show abstract

“…Molecular weight determinations using light scattering techniques were performed on a Dawn F multi-angle laser light-scattering photometer (Wyatt Technology Corporation, Santa Barbara, CA) according to the methodology previously described [6]. An aliquot of sample was manually injected onto a Pharmacia Superose 12 gel filtration column and the scattering intensity and RI signal of the eluting peaks were monitored.…”

Section: Light Scatteringmentioning

confidence: 99%

Lateral self‐assembly of E‐cadherin directed by cooperative calcium binding

Alattia¹,

Ames²,

Porumb³

et al. 1997

FEBS Letters

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“…Here we will illustrate the use of the latter technique. Because several groups have reviewed the method of static light scattering for protein analysis in recent years [5][6][7][8][9][10][11] we will focus specifically on issues related to the analysis of membrane proteins. The relevant theory of static light scattering for membrane protein analysis will be presented, followed by an overview of practical aspects illustrated by a number of examples from our lab.…”

Section: Introductionmentioning

confidence: 99%