Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions

Wen, Jie; Arakawa, Tsutomu; Philo, John S.

doi:10.1006/abio.1996.0345

Cited by 496 publications

(412 citation statements)

References 16 publications

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“…Determination of protein mass content and molecular weight was performed by the ASTRA 6.1 software (Wyatt Technology) using the conjugate analysis algorithm. More detail on the use of three detector systems for the analysis of protein complexes is reported elsewhere 60, 61…”

Section: Methodsmentioning

confidence: 99%

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

et al. 2018

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Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D–K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo‐trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR‐FTIR) reduction in β‐sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α‐helix content increased by 7% (CD) to 13% (ATIR‐FTIR). Maintenance of a native‐like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

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Section: Methodsmentioning

confidence: 99%

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

et al. 2018

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“…The protein concentrations were obtained by amino acid analysis (W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University). The dn/dc value (change of solution refractive index with respect to a change in concentration of the molecules being investigated) is fairly constant for proteins (22) and was set to 0.185 for the analysis of the light-scattering data.…”

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

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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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“…(5) and (5a) to determine the molecular mass of a protein is known as the two-detector method [8,9]. The method can be used to determine the molecular mass of any macromolecule as long as dn/dc is known.…”

Section: Soluble Proteinsmentioning

confidence: 99%

“…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%

Static light scattering to characterize membrane proteins in detergent solution

Slotboom

Duurkens

Olieman

et al. 2008

Methods

167

176

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Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions

Cited by 496 publications

References 16 publications

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

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

Static light scattering to characterize membrane proteins in detergent solution

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