Succinimidyl Residue Formation in Hen Egg-White Lysozyme Favors the Formation of Intermolecular Covalent Bonds without Affecting Its Tertiary Structure

Abstract

Protein chemical degradations occur naturally into living cells as soon as proteins have been synthesized. Among these modifications, deamidation of asparagine or glutamine residues has been extensively studied, whereas the intermediate state, a succinimide derivative, was poorly investigated because of the difficulty of isolating those transient species. We used an indirect method, a limited thermal treatment in the dry state at acidic pH, to produce stable cyclic imide residues in hen lysozyme molecules, enabling us to examine the structural and functional properties of so modified proteins. Five cyclic imide rings have been located at sites directly accessible to solvent and did not lead to any changes in secondary or tertiary structures. However, they altered the catalytic properties of lysozyme and significantly decreased the intrinsic stability of the molecules. Moreover, dimerization occurred during the treatment, and this phenomenon was proportional to the extent of chemical degradation. We propose that succinimide formation could be responsible for covalent bond formation under specific physicochemical conditions that could be found in vivo.

Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: Lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane

“…This improved activity is supposed to be related to the modified physicochemical properties of DH-L. DH-L is more hydrophobic, flexible and surface active than N-L, but its secondary and tertiary structures remain intact [29,30]. It is thus relevant to compare the interaction of native and dry-heated lysozymes with LPS, the lipid components of the outer leaflet of the outer membrane of Gram negative bacteria.…”

“…However, differences in the behavior of DH-L versus N-L with the LPS monolayer can be noticed. This modified behavior could be related to its different physico-chemical properties such as increased hydrophobicity, surface-activity, positive charge and flexibility [29,30].…”

An Arabidopsis ATP-Dependent, DEAD-Box RNA Helicase Loses Activity upon IsoAsp Formation but Is Restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE

“…An enzyme can be replaced through de novo transcription (Li et al, 2001) assuming the whole of the translational apparatus is intact and functional. While this is usually the case, nowhere in the plant is this generalization more jeopardized than in cells of dehydrated seeds (Kushwaha et al, 2013), where spontaneous succinimide formation (the first step in isoAsp formation) but not protein repair is possible in the anhydrous environment (Desfougères et al, 2011). Upon imbibition (rehydration), succinimide rapidly and spontaneously hydrolyzes to either Asp (;15 to 30%) or isoAsp (;85 to 70%).…”

“…This capacity forms the basis of agriculture, allowing a portion of harvested seed to be retained to produce next season's crop. In this partially dehydrated state, proteins can undergo spontaneous degradation to form the succinimide derivative of Asn and Asp residues (Desfougères et al, 2011) that is then largely converted to the isomerized isoAsp derivative upon imbibition. IsoAsp formation is often detrimental to proper protein function and, if unrepaired, can be lethal (Ogé et al, 2008;Verma et al, 2013).…”

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics

“…The nucleophilic functional group with free amine in the chemical molecule like hydrazines and hydroxylamines can potentially react with the cyclic succinimide intermediate formed as part of degradation pathway in the asparagine residue with all the possible factors leading to a covalent adduct [35,[38][39][40][41][42][43][44][45][46]. Tris with its free amine, a potential nucleophilic functional group, reacts with the cyclic asparagine-succinimide intermediate leading to the formation of in-solution covalent adduct that gives the mass of 104 Da addition at the asparagine site adjacent to the glycine residue (Scheme 1).…”