Mass spectrometric methods to analyze the structural organization of macromolecular complexes

Rajabi, Khadijeh; Ashcroft, Alison E.; Radford, Sheena E.

doi:10.1016/j.ymeth.2015.03.004

Cited by 61 publications

(44 citation statements)

References 113 publications

(120 reference statements)

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“…We used HDX-MS to compare the hydrogen/ deuterium exchange profiles for the optimally purified gp140 proteins of both designs, a technique that measures the relative solvent accessibility of different regions of proteins (71,72). Because of sequence variation between the constructs, only a few pepsin-generated peptides could be directly compared between all four 92UG037.8 and CZA97.012 Env proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

166

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We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140 UNC -Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41 ECTO ) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41 ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ϳ20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140 UNC -Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components. IMPORTANCESoluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins. R ecombinant, soluble envelope glycoprotein (Env) gp140 trimers from...

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Section: Resultsmentioning

confidence: 99%

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Ringe

Yasmeen

Ozorowski

et al. 2015

J Virol

166

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“…These span the range from hydrogen/deuterium exchange mass spectrometry, chemical surface labeling techniques, cross-linking mass spectrometry, up to the direct mass analysis of intact protein assemblies [9]. The latter arose following initial work by a few laboratories in the 1990s [10, 11], demonstrating that noncovalent interactions could be preserved in the gas phase for analysis, enabling information on subunit stoichiometry, binding partners, protein complex topology, protein dynamics, and even binding affinities from a single mass spectrometric analysis [12–17]. Cumulatively, these endeavours have been later coined by the term “native mass spectrometry,” which now has become a commonly accepted term for these activities as evidenced also by the name of the 31st ASMS Asilomar Conference, “Native Mass Spectrometry-Based Structural Biology” held in the autumn of 2015.…”

Section: Introductionmentioning

confidence: 99%

Native Mass Spectrometry: What is in the Name?

Leney

Heck

2016

J. Am. Soc. Mass Spectrom.

499

480

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Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstractᅟ

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“…These approaches label most of the amino acids to illuminate protein-protein, and protein-ligand interactions. 15 HDX is widely used for soluble proteins, and the extent of exchange reports on H bonding and solvent accessibility of the protein backbone. 16–17 HDX can be effective for membrane proteins when conducted in the presence of detergent micelles provided the protocol includes fast isolation, good digestion efficiency, and solubility of materials.…”

Section: Introductionmentioning

confidence: 99%

Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc

et al. 2016

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Although membrane proteins are crucial participants in photosynthesis and other biological processes, many lack high-resolution structures. Prior to achieving a high-resolution structure, we are investigating whether MS-based footprinting can provide coarse-grained protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding. Our platform probes topology and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochemical oxidation of proteins (FPOP), and lipid Nanodiscs, which more similar to the native membrane environment than are the widely used detergent micelles. We describe here results that show a protein’s outer membrane regions are more heavily footprinted by OH radicals whereas the regions spanning the lipid bilayer remain inert to the labeling. Nanodiscs generally exhibit more protection of membrane proteins compared to detergent micelles and less shielding to those protein residues that exist outside the membrane. The combination of immobilizing the protein in Nanodiscs and footprinting with the FPOP approach is a feasible approach to map extra-membrane protein surfaces, even at the amino-acid level, and to illuminate intrinsic membrane protein topology.

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Mass spectrometric methods to analyze the structural organization of macromolecular complexes

Cited by 61 publications

References 113 publications

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Native Mass Spectrometry: What is in the Name?

Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc

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