Different Equilibrium Stability Behavior of ScFv Fragments:  Identification, Classification, and Improvement by Protein Engineering

Wörn, Arne; Plückthun, Andreas

doi:10.1021/bi9902079

Cited by 128 publications

(97 citation statements)

References 30 publications

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“…A 2-state denaturation model, which assumes an equilibrium between folded and denatured forms, did not provide adequate agreement with the data. This is consistent with other studies on scFv proteins, which indicate multiple unfolding transitions [98]. The free energy of unfolding cannot therefore be determined from the fluorescence profiles; instead we report the denaturant concentration at the midpoint of the fluorescence change (referred to as C mid ) in Table 1 to provide a measure of the relative stabilities.…”

Section: Overcharged Mutants Exhibit Lower Conformational Stabilitiessupporting

confidence: 82%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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a b s t r a c tThe aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

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Section: Overcharged Mutants Exhibit Lower Conformational Stabilitiessupporting

confidence: 82%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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“…To a first approximation, the residues on the Ab-Ag interface determine specificity and affinity, whereas those in the core partially determine stability. Mutations at the interface between the two variable domains will also contribute to stability (24) and binding affinity (25) and may contribute to the relative orientation of the domains. Surface mutations outside the interfaces may affect solvation properties and aggregation propensity but are expected to have little effect as long as polar surface area is maintained.…”

Section: Region-specific Mutation Type and Locationmentioning

confidence: 99%

Trends in Antibody Sequence Changes during the Somatic Hypermutation Process

Clark

Ganesan

Papp

et al. 2006

The Journal of Immunology

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Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are ∼4% for core residues, ∼5% for surface residues, and ∼12% for residues in common Ag-contacting positions, excepting the those coded by the D gene.

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“…In addition, thermodynamic stability is usually compromised because the stability of most V H domains is augmented by contacts at the light chain interface (13). The elucidation of a number of camelid V H H domain structures has provided insights into how these challenges to structural integrity are accommodated (6, 14 -19).…”

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confidence: 99%

Comprehensive Analysis of the Factors Contributing to the Stability and Solubility of Autonomous Human VH Domains

Barthelemy¹,

Raab²,

Appleton³

et al. 2008

Journal of Biological Chemistry

161

152

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We report a comprehensive analysis of sequence features that allow for the production of autonomous human heavy chain variable (V H ) domains that are stable and soluble in the absence of a light chain partner. Using combinatorial phage-displayed libraries and conventional biophysical methods, we analyzed the entire former light chain interface and the third complementarity determining region (CDR3). Unlike the monomeric variable domains of camelid heavy chain antibodies (V H H domains), in which autonomous behavior depends on interactions between the hydrophobic former light chain interface and CDR3, we find that the stability of many in vitro evolved V H domains is essentially independent of the CDR3 sequence and instead derives from mutations that increase the hydrophilicity of the former light chain interface by replacing exposed hydrophobic residues with structurally compatible hydrophilic substitutions. The engineered domains can be expressed recombinantly at high yield, are predominantly monomeric at high concentrations, unfold reversibly, and are even more thermostable than typical camelid V H H domains. Many of the stabilizing mutations are rare in natural V H and V H H domains and thus could not be predicted by studying natural sequences and structures. The results demonstrate that autonomous V H domains with structural properties beyond the scope of natural frameworks can be derived by using non-natural mutations, which differ from those found in camelid V H H domains. These findings should enable the development of libraries of synthetic V H domains with CDR3 diversities unconstrained by structural demands.It was long believed that all antibodies are composed of heavy and light chains and consequently that the simplest antigenbinding unit is the variable fragment (Fv), 2 a heterodimer of heavy and light chain variable domains (V H and V L , respectively) (1). This supposition has held true for most species, but members of the Camelidae family have proven to be notable exceptions (2, 3). Camelids produce not only conventional antibodies but also "heavy chain antibodies" that are devoid of light chains (4). Consequently, the antigen-binding site of a heavy chain antibody is contained within a monomeric variable domain (V H H, for heavy chain variable domain of a heavy chain antibody), which has evolved to be autonomously stable in the absence of a light chain partner.The simplicity of the V H H domain architecture has attracted interest from protein engineers concerned with developing novel antibodies for biotechnological and therapeutic applications. V H H domains are well suited for use as modular building blocks that can be linked together to assemble multivalent or bispecific antibodies (5). The third complementarity determining loop (CDR3) of camelid V H H domains is often unusually long, and it has been suggested that these protruding loops may be especially effective for targeting active site clefts (6) and cryptic viral epitopes (7). Furthermore, because of their small size, V H H domains are r...

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Different Equilibrium Stability Behavior of ScFv Fragments: Identification, Classification, and Improvement by Protein Engineering

Cited by 128 publications

References 30 publications

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Trends in Antibody Sequence Changes during the Somatic Hypermutation Process

Comprehensive Analysis of the Factors Contributing to the Stability and Solubility of Autonomous Human VH Domains

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