A kinetic trap is an intrinsic feature in the folding pathway of single-chain Fv fragments

Hoyer, Wolfgang; Ramm, Kathrin; Plückthun, Andreas

doi:10.1016/s0301-4622(02)00022-4

Cited by 18 publications

(10 citation statements)

References 20 publications

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“…These results suggest that the presence of the wild-type Vα domain at the N-terminus prevented the Vβ (B7) domain from folding. Indeed, the influence of the stability of one V domain on the folding of another V domain has been demonstrated in the analagous scFv system (Hoyer et al, 2002). …”

Section: Resultsmentioning

confidence: 99%

Structural features of T cell receptor variable regions that enhance domain stability and enable expression as single-chain VαVβ fragments

Richman

Aggen

Dossett

et al. 2009

Molecular Immunology

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The variable (V) domains of antibodies and T cell receptors (TCRs) share sequence homology and striking structural similarity. Single-chain antibody V domain constructs (scFv) are routinely expressed in a variety of heterologous systems, both for production of soluble protein as well as for in vitro engineering. In contrast, single-chain T cell receptor V domain constructs (scTCR) are prone to aggregation and misfolding and are refractory to display on phage or yeast in their wild-type form. However, through random mutagenesis and yeast display engineering, it has been possible to isolate scTCR mutants that are properly folded and displayed on the yeast surface. These displayed mutants can serve not only as a scaffold for further engineering but also as scTCR variants that exhibit favorable biophysical properties in E. coli expression. Thus, a more comprehensive understanding of the V domain mutations that allowed display would be beneficial. Our goal here was to identify generalizable patterns of important mutations that can be applied to different TCRs. We compared five different scTCRs, four from mice and one from a human, for yeast surface display. Analysis of a collection of mutants revealed four distinct regions of TCR V domains that were most important for enabling surface expression: the Vα-Vβ interface, the HV4 of Vβ, and the region of the Vα and Vβ domains normally apposed against the constant (C) domains. Consistent with the role of the V-C interface in surface display, reconstitution of this interface, by including the constant domains of each chain, allowed V domain display and αβ chain association on the yeast surface, thus providing an alternative TCR scaffold. However, the surface levels of TCR achieved with engineered scTCR mutants were superior to that of the VαCα/VβCβ constructs. Therefore, we describe further optimization of the current strategy for surface display of the single-chain format in order to facilitate yeast display engineering of a broader range of scTCRs.

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Section: Resultsmentioning

confidence: 99%

Structural features of T cell receptor variable regions that enhance domain stability and enable expression as single-chain VαVβ fragments

Richman

Aggen

Dossett

et al. 2009

Molecular Immunology

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“…[41][42][43] In natural proteins this extreme behavior is not observed, although off-pathway intermediates have been characterized for several proteins. [44][45][46][47][48] Intermediate states may induce irreversible aggregation, often mediated by hydrophobic interactions or by the formation of an amyloid beta structure. 49,50 Molecular chaperones assist folding in vivo by sequestrating proteins in misfolded and intermediate states, thus preventing aggregation and speeding up the folding process.…”

Section: Unfolding Versus Misfoldingmentioning

confidence: 99%

Genomic Determinants of Protein Folding Thermodynamics in Prokaryotic Organisms

Bastolla

Moya

Viguera

et al. 2004

Journal of Molecular Biology

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“…In some cases, two-state folding behavior has been reported; 31 in others, folding via a highly structured intermediate 27,29,30 or a misfolded species was assumed. 35 These differences do of course not rule out a conserved folding mechanism. They might rather represent variations of a motif due to sequence differences which influence the stabilities of different populated species.…”

Section: Introductionmentioning

confidence: 99%

Influence of the Internal Disulfide Bridge on the Folding Pathway of the CL Antibody Domain

Feige

Hagn

Esser

et al. 2007

Journal of Molecular Biology

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A kinetic trap is an intrinsic feature in the folding pathway of single-chain Fv fragments

Cited by 18 publications

References 20 publications

Structural features of T cell receptor variable regions that enhance domain stability and enable expression as single-chain VαVβ fragments

Structural features of T cell receptor variable regions that enhance domain stability and enable expression as single-chain VαVβ fragments

Genomic Determinants of Protein Folding Thermodynamics in Prokaryotic Organisms

Influence of the Internal Disulfide Bridge on the Folding Pathway of the CL Antibody Domain

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