Intra-molecular cross-linking of acidic residues for protein structure studies.

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Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the ... continued below

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24 p.

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Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr & Schoeniger, Joseph S. March 1, 2005.

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Description

Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target of choice will yield sufficient distance constraints to develop a structural model.

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24 p.

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  • Report No.: SAND2005-1698
  • Grant Number: AC04-94AL85000
  • DOI: 10.2172/922769 | External Link
  • Office of Scientific & Technical Information Report Number: 922769
  • Archival Resource Key: ark:/67531/metadc901866

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  • March 1, 2005

Added to The UNT Digital Library

  • Sept. 27, 2016, 1:39 a.m.

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  • Dec. 7, 2016, 3:45 p.m.

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Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr & Schoeniger, Joseph S. Intra-molecular cross-linking of acidic residues for protein structure studies., report, March 1, 2005; United States. (digital.library.unt.edu/ark:/67531/metadc901866/: accessed November 20, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.