Determining Protein−Protein Interactions by Oxidative Cross-Linking of a Glycine-Glycine-Histidine Fusion Protein † (original) (raw)
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Novel inter‐protein cross‐link identified in the GGH‐ecotin D137Y dimer
Protein Science, 2009
In the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl–glycyl–histidine (GGH) mediates efficient and specific oxidative protein cross‐linking. The fusion of GGH to the N terminus of a protein allows for the cross‐linking reagent to be delivered in a site‐specific fashion, making this system extremely useful for analyzing protein–protein contacts in complicated mixtures of biomolecules. Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross‐linking efficiency. Cross‐linking increased four‐fold for GGH‐ecotin D137Y in comparison to wild‐type GGH‐ecotin, presumably through bityrosine formation at the dimer interface. Here we report the first complete structural analysis of the cross‐linked GGH‐ecotin D137Y dimer. Using a combination of mass spectrometric ...
Enzyme-catalyzed protein crosslinking
Applied Microbiology and Biotechnology, 2013
The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.
Chemically Mediated Site-Specific Proteolysis. Alteration of Protein−Protein Interaction †
Biochemistry, 2002
The design and synthesis of a novel iodine-labile serine protease inhibitor was realized by the use of an ecotin analogue containing allylglycine at position 84 in lieu of methionine. Allylglycinecontaining ecotins were synthesized by in vitro translation of the ecotin gene containing an engineered nonsense codon (TAG) at the positions of interest. A misacylated suppressor tRNA activated with the unnatural amino acid allylglycine was employed for the suppression of the nonsense codons in a cell-free protein biosynthesizing system, permitting the elaboration of ecotin analogues containing allyglycine at the desired sites. The derived ecotin analogues were capable of inhibiting bovine trypsin with inhibitory constants (K i s) comparable to that of wild-type ecotin. Iodine treatment of ecotin analogue Met84 A Gly resulted in the deactivation of ecotin, caused by peptide backbone cleavage at its P1 reactive site. Upon iodine treatment, active trypsin could be released from the protein complex with ecotin analogue Met84 A Gly. This constitutes a novel strategy for modulation of serine protease activity and more generally for alteration of protein-protein interaction by a simple chemical reagent.
Insights on Chemical Crosslinking Strategies for Proteins
Molecules
Crosslinking of proteins has gained immense significance in the fabrication of biomaterials for various health care applications. Various novel chemical-based strategies are being continuously developed for intra-/inter-molecular crosslinking of proteins to create a network/matrix with desired mechanical/functional properties without imparting toxicity to the host system. Many materials that are used in biomedical and food packaging industries are prepared by chemical means of crosslinking the proteins, besides the physical or enzymatic means of crosslinking. Such chemical methods utilize the chemical compounds or crosslinkers available from natural sources or synthetically generated with the ability to form covalent/non-covalent bonds with proteins. Such linkages are possible with chemicals like carbodiimides/epoxides, while photo-induced novel chemical crosslinkers are also available. In this review, we have discussed different protein crosslinking strategies under chemical method...
Novel Amidinating Cross-Linker for Facilitating Analyses of Protein Structures and Interactions
Analytical Chemistry, 2010
A novel bifunctional thioimidate cross-linking reagent (diethyl suberthioimidate) that modifies amines without sacrificing their native basicity is developed. Intermolecular cross-linking of neurotensin and intramolecular crosslinking of cytochrome c under physiological conditions is investigated with this reagent. Because it does not perturb the electrostatic properties of a protein, it is unlikely to lead to artifactual conclusions about native protein structure. The interpeptide cross-links formed with this reagent are easily separated from other tryptic fragments using strong cation exchange chromatography, and they have a readily identified mass spectrometric signature. The use of this novel amidinating protein cross-linking reagent holds great promise for efficient, large-scale structural analysis of complex systems.
Covalent cross-linking of proteins without chemical reagents
Protein Science, 2002
A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85°C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.
Strategy for selective chemical cross-linking of tyrosine and lysine residues
Journal of the American Society for Mass Spectrometry, 2004
Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin -chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminustyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH.
FEBS Letters, 1996
Ecotin, a homodimer protein of E. coil, is a unique member of canonical serine proteinase inhibitors, since it is a potent agent against a variety of serine proteinases having different substrate specificity. Monomers of ecotin are held together mostly by their long C-terminal strands that are arranged as a two-stranded antiparallel ~-sheet in the functional dimer. One ecotin dimer can chelate two proteinase molecules, each of them bound to both subunits of ecotin at two different sites, namely the specific primary and the non-specific secondary binding sites. In this study the genes of wild type ecotin and its MetS4Arg P1 site mutant were truncated resulting in new forms of ecotin that lack 10 amino acid residues at their C-terminus. These mutants do not dimerize spontaneously, though in combination with trypsin they assemble into the familiar heterotetramer. Our data suggest that this heterotetramer exists even in extremely diluted solutions, and the interaction, which is responsible for the dimerization of ecotin, contributes to the stability of the heterotetrameric complex.
An amino acid-based heterofunctional cross-linking reagent
Amino Acids, 2014
We describe the synthesis and characterization of a new lysine-based heterofunctional cross-linking reagent. It carries two readily available aminooxy functionalities and an activated and protected thiol group that is capable of generating reducible disulfides, the former enable bioorthogonal modification of ketones and aldehydes by the formation of an oxime bond. The efficacy of the linker was proven by coupling two doxorubicin molecules to the functionalized amino acid core and the subsequent bioconjugation of this drug conjugate with a thiolated antibody.