[11] General principles for photoaffinity labeling of peptide hormone receptors

Neberle, Alex; Graan, P.N.E. de

doi:10.1016/0076-6879(85)09081-4

Cited by 47 publications

(17 citation statements)

References 108 publications

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“…The presence of DTT renders an unknown amount of the p-APB modified prSS incapable of cross-linking, but still able to bind to the receptor, thereby lowering the yield of conjugate. Finally, upon generation of the nitrene by UV light, it is possible for the nitrene to react with the MOPS buffer, sorbitol, or membrane lipids instead of the receptor protein (7), still further eroding the efficiency of crosslinking. We are attempting to overcome these problems by using plastocyanin and ferrodoxin precursors and by using modifications of the cross-linking reaction conditions.…”

Section: Ss -mentioning

confidence: 99%

“…It should be possible to examine precursor/ receptor interactions and to identify the receptor proteins using some of the techniques that have been applied to other receptor/ ligand interactions, such as the well characterized interactions between polypeptide hormones and their receptors. One approach which has been successfully applied to the problem of receptor identification is the use of photoaffinity cross-linkers (4,7,12,13). Photoaffinity cross-linkers can be sequentially controlled and therefore are more specific than homobifunctional cross-linkers (7,12 Chloroplast Isolation.…”

mentioning

confidence: 99%

“…One approach which has been successfully applied to the problem of receptor identification is the use of photoaffinity cross-linkers (4,7,12,13). Photoaffinity cross-linkers can be sequentially controlled and therefore are more specific than homobifunctional cross-linkers (7,12 Chloroplast Isolation. The peas were grown and chloroplasts isolated as previously described by Cline et al (5) with the following modifications.…”

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confidence: 99%

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Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase

Cornwell¹,

Keegstra²

1987

Plant Physiol.

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Most chloroplast proteins are encoded by nuclear genes and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported posttranshationally into the chloroplasts, where they are proteolytically processed, and sorted to their proper locations. The first step of this import process is thought to be the binding of precursors to putative receptors on the outer envelope membrane of chloroplasts. We have investigated the interaction of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with its putative receptor by using a heterobifunctional, photoactivatable cross-linker. The resulting cross-linked conjugate has a molecular weight of 86,000, and is present on the surface of chloroplasts as determined by its sensitivity to digestion with protease. Control experiments demonstrated that the label in the conjugate is derived from small subunit precursor and that the conjugate is formed only when modified precursor is reacted in the presence of chloroplasts. Based on these results, we postulate that a protein on the surface of chloroplasts is part of the receptor which interacts with the small subunit precursor.Most proteins found in chloroplasts and mitochondria are nuclear encoded and synthesized on cytoplasmic ribosomes. The majority of these proteins are made as higher mol wt precursors which are then imported into the respective organelle, proteolytically processed to the mature size and sorted to their proper location (cf 9 and 21 for recent reviews). The first step of this import process is thought to be the recognition and binding of the precursor protein to a specific receptor on the surface of the proper organelle. The most extensive evidence for the involvement ofspecific receptors comes from the study of mitochondrial protein import (9). One type of evidence for the involvement of mitochondrial surface proteins comes from the inhibition of protein import by pretreatment of mitochondria with proteases (19,23,24 Despite the studies described above, we still know very little about the putative receptor proteins or their role in the import process. For example, a receptor protein has not been identified in either mitochondria or chloroplasts. The lack of rigorous proof for the presence of receptors, combined with the observation that precursors can interact with lipids (20), has led to the suggestion that precursor proteins may interact directly with lipid bilayer membranes without the involvement of proteinaceous receptors (20,22). Thus, it becomes important to determine whether proteinaceous receptors exist, to identify the proteins which function as receptors, and to investigate their involvement in the import process. It should be possible to examine precursor/ receptor interactions and to identify the receptor proteins using some of the techniques that have been applied to other receptor/ ligand interactions, such as the well characterized interactions between polypeptide hormones and their receptors. One approach which has been successfull...

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Section: Ss -mentioning

confidence: 99%

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confidence: 99%

mentioning

confidence: 99%

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Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase

Cornwell¹,

Keegstra²

1987

Plant Physiol.

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“…In dimers and hexamers, Phe B24 and Phe B25 also participate in an intermolecular ␤-sheet, an essential element of insulin's storage form in the ␤-cell (3). Whereas considerable evidence indicates that the exposed side chain of Phe B25 contacts the insulin receptor (6, including previous photo crosslinking studies [7,8]), the roles of Val A3 and Phe B24 have long been the subject of speculation (3,9 -13).To test whether residues A3, B24, and B25 contact the insulin receptor, we have synthesized insulin analogs containing a photo-activatable derivative of phenylalanine (Phe), para-azido-Phe (Pap) (8,14). Pap was chosen based on its rigidity and small size (relative to other photoactivable moieties), thus limiting the distance range for cross-linking.…”

mentioning

confidence: 99%

“…To test whether residues A3, B24, and B25 contact the insulin receptor, we have synthesized insulin analogs containing a photo-activatable derivative of phenylalanine (Phe), para-azido-Phe (Pap) (8,14). Pap was chosen based on its rigidity and small size (relative to other photoactivable moieties), thus limiting the distance range for cross-linking.…”

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confidence: 99%

Diabetes-Associated Mutations in Insulin Identify Invariant Receptor Contacts

Chen

Wang

et al. 2004

Diabetes

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Mutations in human insulin3 Leu. By analogy to the nomenclature describing abnormal hemoglobins, these are designated insulins Wakayama, Los Angeles, and Chicago, respectively (1). We demonstrate here that these mutations occur at contact sites between insulin and the ␣-subunit of the insulin receptor.The structure of insulin is well characterized by crystallography (3) and nuclear magnetic resonance spectroscopy (4,5) (Fig. 1A). Residues A3, B24, and B25 exhibit distinct environments. Whereas Phe B25 projects from the surface, Val A3 and Phe B24 are engaged in long-range interactions (Fig. 2). Val A3 contacts Tyr B26 and Pro B28 at an interface between the NH 2 -terminal A-chain ␣-helix and COOH-terminal B-chain ␤-strand (Fig. 2B) , Tyr B16 , and Cys B19 to stabilize the supersecondary structure of the B-chain. In dimers and hexamers, Phe B24 and Phe B25 also participate in an intermolecular ␤-sheet, an essential element of insulin's storage form in the ␤-cell (3). Whereas considerable evidence indicates that the exposed side chain of Phe B25 contacts the insulin receptor (6, including previous photo crosslinking studies [7,8]), the roles of Val A3 and Phe B24 have long been the subject of speculation (3,9 -13).To test whether residues A3, B24, and B25 contact the insulin receptor, we have synthesized insulin analogs containing a photo-activatable derivative of phenylalanine (Phe), para-azido-Phe (Pap) (8,14). Pap was chosen based on its rigidity and small size (relative to other photoactivable moieties), thus limiting the distance range for cross-linking. Modified A-and B-chains were prepared by solid-phase synthesis using the photostable precursor para-amino-Phe. To enable efficient detection of crosslinked peptides, the ␣-amino group of the B-chain was biotinylated (8). The nonstandard side chain was introduced into an engineered insulin monomer (DKP-insulin, which contains three B-chain substitutions: His B10 3 Asp, Pro B28 3 Lys, and Lys B29 3 Pro), chosen as a template for its efficiency of synthesis, enhanced receptor binding, and absence of confounding self-association (4). A3, B24, and B25 para-amino-Phe analogs exhibit respective receptorbinding affinities of 2.0 Ϯ 0.2, 59 Ϯ 2, and 147 Ϯ 3% relative to native insulin (K d 0.48 nmol/l); the affinity of the biotin adduct of DKP-insulin is 132 Ϯ 5% (assays performed in triplicate). Corresponding analogs were prepared at terminal positions A1 and A21 (ordinarily conserved as Gly and Asn, respectively) at the periphery of insulin's putative receptor-binding surface (3,15); their relative receptorbinding affinities are 46 Ϯ 5 and 79 Ϯ 22%. Asn A21 projects from the surface near Gly B23 and Phe B25 ( Fig. 2A). Conversion of para-amino-Phe to Pap in the intact hormones was verified by mass spectrometry.

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Analysis of Protein Ligand‐Receptor Binding by Photoaffinity Cross‐Linking

2015

CP Protein Science

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Photoaffinity cross-linking is a rapidly developing technology for studying biomolecular interactions, including protein ligand-receptor binding. This technology provides detailed binding information including receptor contact sites, active conformation of receptor-ligand complexes, global binding surfaces, and binding modes. Advancements in genetic technology have enabled non-natural photoactive amino acid derivatives to be incorporated into designer or target proteins, providing a host of new opportunities for manufacturing protein photo-probes while bypassing the traditional peptide or small protein limits of classical chemical synthesis. This unit provides several protocols for performing basic photoaffinity cross-linking and related analyses for applications in ligand-receptor binding and protein-protein interactions.

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[11] General principles for photoaffinity labeling of peptide hormone receptors

Cited by 47 publications

References 108 publications

Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase

Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase

Diabetes-Associated Mutations in Insulin Identify Invariant Receptor Contacts

Analysis of Protein Ligand‐Receptor Binding by Photoaffinity Cross‐Linking

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