Antibody Fragments for Stabilization and Crystallization of G Protein-Coupled Receptors and Their Signaling Complexes

Shukla, Arun K.; Gupta, Charu; Srivastava, Ashish; Jaiman, Deepika

doi:10.1016/bs.mie.2015.01.010

Cited by 10 publications

(4 citation statements)

References 30 publications

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“…V H H domains have been shown to engage epitopes generally inaccessible to conventional antibodies on G-protein coupled receptors (GPCRs), which are a class of medically important protein targets. For example, V H Hs have been used as chaperones to facilitate structural analysis of conformational states of membrane proteins [62, 63]. The limited ability of the human antibody response to target such proteins may be due to the limited diversity of CDR loop length, the large size of the antigen-binding units and/or the fact that the paratopes have mostly flat or concave topologies.…”

Section: Introductionmentioning

confidence: 99%

Engineered Autonomous Human Variable Domains

Nilvebrant

Tessier

Sidhu

2017

CPD

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The complex multi-chain architecture of antibodies has spurred interest in smaller derivatives that retain specificity but can be more easily produced in bacteria. Domain antibodies consisting of single variable domains are the smallest antibody fragments and have been shown to possess enhanced ability to target epitopes that are difficult to access using multidomain antibodies. However, in contrast to natural camelid antibody domains, human variable domains typically suffer from low stability and high propensity to aggregate. This review summarizes strategies to improve the biophysical properties of heavy chain variable domains from human antibodies with an emphasis on aggregation resistance. Several protein engineering approaches have targeted antibody frameworks and complementarity determining regions to stabilize the native state and prevent aggregation of the denatured state. Recent findings enable the construction of highly diverse libraries enriched in aggregation-resistant variants that are expected to provide binders to diverse antigens. Engineered domain antibodies possess unique advantages in expression, epitope preference and flexibility of formatting over conventional immunoreagents and are a promising class of antibody fragments for biomedical development.

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Section: Introductionmentioning

confidence: 99%

Engineered Autonomous Human Variable Domains

Nilvebrant

Tessier

Sidhu

2017

CPD

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“…The motivation for developing antibody fragments stems from an improved rate of tumor uptake and intratumoral distribution [142]. Besides clinical applications, mAb and antibody fragments have proved revolutionary in the field of scientific research with applications in protein detection [143], immunohistochemistry [144], ELISA [145] and membrane protein structure determination [146]. Antibody fragments generally minimize conformational heterogeneity and intrinsic flexibility along with improving crystal contacts leading to better X-ray diffraction and high-resolution structure determination of membrane proteins [147,148].…”

Section: Antibody Fragmentsmentioning

confidence: 99%

New Horizons in Structural Biology of Membrane Proteins: Experimental Evaluation of the Role of Conformational Dynamics and Intrinsic Flexibility

2022

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A plethora of membrane proteins are found along the cell surface and on the convoluted labyrinth of membranes surrounding organelles. Since the advent of various structural biology techniques, a sub-population of these proteins has become accessible to investigation at near-atomic resolutions. The predominant bona fide methods for structure solution, X-ray crystallography and cryo-EM, provide high resolution in three-dimensional space at the cost of neglecting protein motions through time. Though structures provide various rigid snapshots, only an amorphous mechanistic understanding can be inferred from interpolations between these different static states. In this review, we discuss various techniques that have been utilized in observing dynamic conformational intermediaries that remain elusive from rigid structures. More specifically we discuss the application of structural techniques such as NMR, cryo-EM and X-ray crystallography in studying protein dynamics along with complementation by conformational trapping by specific binders such as antibodies. We finally showcase the strength of various biophysical techniques including FRET, EPR and computational approaches using a multitude of succinct examples from GPCRs, transporters and ion channels.

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“…Bacterial membrane proteins have also been trapped by conformationspecific nanobodies, both for structural characterization (96,97) and for inhibition of their activity (98). Several accounts and reviews cover the development of nanobodies as reagents for structural studies (10) and their use as chaperones (2,90). A comprehensive overview of structures containing nanobodies was published (99), and a regularly updated database is available online (100).…”

Section: Targeting Membrane Proteinsmentioning

confidence: 99%

Exploring cellular biochemistry with nanobodies

Cheloha

Harmand

Wijne

et al. 2020

Journal of Biological Chemistry

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Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. While known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.

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Antibody Fragments for Stabilization and Crystallization of G Protein-Coupled Receptors and Their Signaling Complexes

Cited by 10 publications

References 30 publications

Engineered Autonomous Human Variable Domains

Engineered Autonomous Human Variable Domains

New Horizons in Structural Biology of Membrane Proteins: Experimental Evaluation of the Role of Conformational Dynamics and Intrinsic Flexibility

Exploring cellular biochemistry with nanobodies

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