Protein stability: a crystallographer's perspective

Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

doi:10.1107/s2053230x15024619

Cited by 215 publications

(154 citation statements)

References 260 publications

(249 reference statements)

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“…However, the DSC unfolding peak of E2ΔTM is relatively broad, with a transition width (ΔT 1/2 ) of 10.6°C, which is nearly twice the ΔT 1/2 of HIV-1 gp140 (46,48). A broad ΔT 1/2 may be indicative of a multistep, noncooperative unfolding transition, suggesting the protein may contain different domains that unfold independently (49,50). Next, to determine the influence of the C-terminal stalk region and the N-terminal HVR1 on the stability of E2, we analyzed the E2c construct that lacks these components.…”

Section: Resultsmentioning

confidence: 99%

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

Kong

Lee

Kadam

et al. 2016

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

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108

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Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8°C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.hepatitis C virus | E2 | CD81-binding site | conformational flexibility | protein dynamics H epatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma, infecting more than 2% of the world's population (1). HCV infection is highly prevalent in developing countries and among marginalized populations, such as injection drug users (IDUs) and prisoners in developed countries (2). Effective direct-acting antiviral (DAA) drugs have been developed recently to curb the advance of HCV (3, 4). Nevertheless, new infections are on the rise among young IDUs in developed countries and along drug-trafficking routes (2, 5, 6). Because DAA treatment is prohibitively expensive and does not prevent reinfection (7,8), an effective vaccine is essential for management of the global HCV epidemic (9-11).Despite the significant public health burden, no effective prophylactic vaccine against HCV has been developed. High genetic variability of the virus is a major barrier, particularly in the E1 and E2 envelope glycoproteins that are the primary neutralizing antibody (NAb) targets. E2, the receptor-binding protein, mediates cell entry by interacting with the tetraspanin CD81 and several other cell surface molecules (12, 13). The crystal structure of the E2 core domain (E2c) consists of a central β-sandwich flanked by "front" and "back" layers (14) (Fig. 1). The CD81-binding site (CD81bs) has been mapped by mutagenesis and electron microscopy (EM) to various elements: a conserved N-terminal...

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Section: Resultsmentioning

confidence: 99%

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

Kong

Lee

Kadam

et al. 2016

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

Self Cite

108

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“…The SPA method relies on the availability of a radiolabeled high-affinity ligand and if no ligands are known which bind with submicromolar affinity then an alternative approach will be necessary. Many other methods are commonly used to measure protein stability such as binding of fluorescent dyes and calorimetry 14 but are low-throughput and are either unable to directly measure function or require large amounts of protein. If the SPA method cannot be used, one alternative high-throughput approach is an FSEC-based Thermostability Assay 15 (FSEC-TS), where the sample is heated followed by separation of the fraction of remaining transporter.…”

Section: Discussionmentioning

confidence: 99%

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to <em>S</em>-citalopram

Coleman

Green

Gouaux

2016

JoVE

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The serotonin transporter is a sodium and chloride-coupled transporter that "pumps" extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI -cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are wellbehaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 Å resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

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“…The quality of crystallographic structures is generally higher under conditions where the protein is thermally stable [68], and extending this to cryo-EM is being explored in approaches such as the “Proteoplex” screen devised by Stark and colleagues [17]. The use of surface modification to enhance the proportion of protein complexes that distribute into the “holes” of holey carbon grids is another strategy that helps offset preferential binding of protein complexes to carbon film substrates [69].…”

Section: Grid Modification Methods For Specimen Preparationmentioning

confidence: 99%

Cryo-EM: beyond the microscope

Earl

Falconieri

Milne

et al. 2017

Current Opinion in Structural Biology

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The pace at which cryo-EM is being adopted as a mainstream tool in structural biology has continued unabated over the past year. Initial successes in obtaining near-atomic resolution structures with cryo-EM were enabled to a large extent by advances in microscope and detector technology. Here, we review some of the complementary technical improvements that are helping sustain the cryo-EM revolution. We highlight advances in image processing that permit high resolution structure determination even in the presence of structural and conformational heterogeneity. We also review selected examples where biochemical strategies for membrane protein stabilization facilitate cryo-EM structure determination, and discuss emerging approaches for further improving the preparation of reliable plunge-frozen specimens.

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Protein stability: a crystallographer's perspective

Cited by 215 publications

References 260 publications

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to <em>S</em>-citalopram

Cryo-EM: beyond the microscope

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