Engineered synthetic antibodies as probes to quantify the energetic contributions of ligand binding to conformational changes in proteins

Mukherjee, Somnath; Griffin, Dionne H.; Horn, James R.; Rizk, Shahir S.; Nocula‐Ługowska, Małgorzata; Malmqvist, Magnus; Kim, Sangwoo S.; Kossiakoff, Anthony A.

doi:10.1074/jbc.ra117.000656

Cited by 28 publications

(20 citation statements)

References 33 publications

(33 reference statements)

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“…The binding of sAB-K29 to K29-linked di-ubiquitin was verified by multi-point ELISA (Dominik et al, 2016;Mukherjee et al, 2018) and surface plasmon resonance (SPR). ELISA were performed using plates prepared as above.…”

Section: Fab Binding Characterizationmentioning

confidence: 99%

“…After optimization of the amount of immobilized ligands based on response units (RUs), a series of sAB-K29 concentrations was injected into both the experimental and reference channels. sAB-K29 was injected over a 60s-association phase, followed by a 600s dissociation time (Mukherjee et al, 2018). Following each injection, the chip surface was regenerated using 350 mM EDTA and 50 mM NaOH, and ligands were subsequently immobilized for the following injection.…”

Section: Fab Binding Characterizationmentioning

confidence: 99%

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K29-Linked Ubiquitin Signaling Regulates Proteotoxic Stress Response and Cell Cycle

Yu¹,

Zheng

Erramilli³

et al. 2020

Preprint

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Protein ubiquitination, one of the most common posttranslational modifications, is involved in numerous cellular processes. It shows remarkable topological and functional diversity, a phenomenon known as the ubiquitin code, through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understanding the physiology of the cell. Among the eight possible linkages, K29-linked polyubiquitin is a relatively abundant type of polyubiquitin in both human and yeast cells (Tsuchiya et al., 2018). Despite previous studies, the lack of specific binders as tools to detect K29-linked polyubiquitin has prevented further understanding of its function. In this study, we screened and characterized a synthetic antigen-binding fragment (named sAB-K29) that can specifically recognize K29-linked polyubiquitin. We determined the crystal structure of sAB-K29 bound to K29-linked diubiquitin (diUb), which revealed the molecular basis of sAB-K29 specificity. Using sAB-K29 as a tool, we identified the involvement of K29-linked ubiquitination in RNA processing, the proteotoxic stress response and cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody at the telophase of mitosis and that downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

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Section: Fab Binding Characterizationmentioning

confidence: 99%

Section: Fab Binding Characterizationmentioning

confidence: 99%

K29-Linked Ubiquitin Signaling Regulates Proteotoxic Stress Response and Cell Cycle

Yu¹,

Zheng

Erramilli³

et al. 2020

Preprint

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“…Data were collected at 24-ID-C at the Advanced Photon Source and processed with XDS 41 . Initial phases were determined in Phaser 42 by molecular replacement using previously determined crystal structures of BRIL (PDB code: 4EIY) 22 and sAB (PDB code: 5BJZ) 17 without CDRs as models. Pieces of variable and the constant domains of sABs were used as different ensembles during molecular replacement.…”

Section: Methodsmentioning

confidence: 99%

“…Fabs have been generated by traditional hybridoma approaches, but generally recombinant-based library display methods, such as phage, yeast or ribosome display are more broadly used [13][14][15] . These display methods allow for the flexibility to tune affinities, epitopes and conformational state 16,17 . However, the use of Fabs remains restricted because they have to be produced for each individual system.…”

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confidence: 99%

Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

et al. 2020

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We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

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“…The authors declare that they have no conflicts of interest with the contents of this article. 1 To whom correspondence should be addressed.…”

mentioning

confidence: 99%

Comment on the calculations in protein thermodynamics

Kang

Brooks

2018

Journal of Biological Chemistry

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Engineered synthetic antibodies as probes to quantify the energetic contributions of ligand binding to conformational changes in proteins

Cited by 28 publications

References 33 publications

K29-Linked Ubiquitin Signaling Regulates Proteotoxic Stress Response and Cell Cycle

K29-Linked Ubiquitin Signaling Regulates Proteotoxic Stress Response and Cell Cycle

Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

Comment on the calculations in protein thermodynamics

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