Kinetic and thermodynamic control of the relative yield of the deamidation of asparagine and isomerization of aspartic acid residues

Capasso, Sante; Cerbo, Paola Di

doi:10.1034/j.1399-3011.2000.00778.x

Cited by 26 publications

(21 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This calculation showed that 1.1-1.8 Asp/Asn residues were racemised per polypeptide in a 70-year-old person (Table 1). This factor alone is likely to lead to significant protein denaturation, especially since the process which leads to racemisation of Asp via a cyclic intermediate can also generate three isoaspartyl residues for each altered Asp residue (Capasso and Di Cerbo 2000). The values shown in Table 1 illustrate the quantitative importance of this process for the aged lens; however, it should be noted that different proteins, and regions within them, very likely behave differently in terms of their susceptibility to racemisation, so the values must be considered as estimates only.…”

Section: Discussionmentioning

confidence: 99%

Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses

Hooi

Truscott

2010

AGE

105

122

View full text Add to dashboard Cite

Astract Several amino acids were found to undergo progressive age-dependent racemisation in the lifelong proteins of normal human lenses. The two most highly racemised were Ser and Asx. By age 70, 4.5% of all Ser residues had been racemised, along with >9% of Asx residues. Such a high level of inversion, equivalent to between 2 and 3 D -amino acids per polypeptide chain, is likely to induce significant denaturation of the crystallins in aged lenses. Thr, Glx and Phe underwent age-dependent racemisation to a smaller degree. In model experiments, D -amino acid content could be increased simply by exposing intact lenses to elevated temperature. In cataract lenses, the extent of racemisation of Ser, Asx and Thr residues was significantly greater than for agematched normal lenses. This was true, even for cataract lenses removed from patients at the earliest ages where age-related cataract is observed clinically. Racemisation of amino acids in crystallins may arise due to prolonged exposure of these proteins to ocular temperatures and increased levels of racemisation may play a significant role in the opacification of human lenses.

show abstract

Section: Discussionmentioning

confidence: 99%

Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses

Hooi

Truscott

2010

AGE

105

122

View full text Add to dashboard Cite

show abstract

“…15,16 The relative proportions of aspartyl to isoaspartyl products is under kinetic and thermodynamic control. 17 Based on a large number of model pentapeptide deamidation kinetics, it is well understood that the rate of deamidation of asparagine is significantly determined by the neighbouring amino acids especially the carboxyl side (n11) amino acid.…”

Section: Introductionmentioning

confidence: 99%

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

2014

View full text Add to dashboard Cite

At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75 C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90 C. MutC has retained more than 50% activity even after incubation at 100 C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.

show abstract

“…Radioactive methods utilize the enzyme protein isoaspartyl methyl transferase (PIMT) to selectively methylate isoAsp residues in peptides and proteins with isotopically labeled methyl groups, thus detecting deamidation, but not localizing the site or determining the relative abundance of the products [24,25]. Separation of proteins from their deamidated counterparts can be done by ion-exchange HPLC [23,26,27] or IEF gel electrophoresis [14,28] because there is a net change in the isoelectric point with deamidation. However, the methods are not used for differentiation and relative quantitation of the deamidation products because the acidity constants of the isomers are so similar; isoAsp and Asp were shown to have a difference of only 0.7 pK a units [17].…”

mentioning

confidence: 99%

“…Hydrolysis of the succinimide intermediate in linear peptides typically results in an approximate 3:1 ratio of products favoring the isoAsp form [15][16][17][18][19][20][21], but ratios from succinimide hydrolysis within a structurally restricted environment, such as within a protein's tertiary structure, can deviate from this ratio [22]. For example, N67 of ribonuclease A ultimately deamidated to a 3:2 ratio in favor of the Asp form due to the restricted flexibility of the region by two flanking disulfide bonds [23]. Therefore, a method that determines the relative abundance of the products is necessary for complete characterization of asparaginyl deamidation and Asp isomerization that is suspected to affect a protein's structure and function.…”

mentioning

confidence: 99%

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Cournoyer

Lin

Bowman

et al. 2007

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Relative quantitation of aspartyl and isoaspartyl residue mixtures from asparagine deamidation is demonstrated using electron capture dissociation without prior HPLC separation. The method utilizes the linear relationship found between the relative abundance of the isoaspartyl diagnostic ion, z n -57, and % isoaspartyl content based on the ECD spectra of known isoaspartyl/aspartyl mixtures of synthetic peptides. The observed linearity appears to be sequence independent because the relationship exists despite sequence variations and changes in backbone fragment abundances when isoaspartyl and aspartyl residues are interchanged. Furthermore, a new method to calculate the relative abundances of isomer from protein deamidation without synthetic peptides is proposed and tested using a linear peptide released by protein digestion that contains the deamidation site. The proteolytic peptide can be rapidly aged to the expected 3:1 (isoaspartyl:aspartyl) mixture to generate a two-point calibration standard for ECD analysis. The procedure can then be used to determine the relative abundance of deamidation products from in vivo or in vitro protein aging experiments. (J Am Soc Mass Spectrom 2007, 18, 48 -56)

show abstract

Kinetic and thermodynamic control of the relative yield of the deamidation of asparagine and isomerization of aspartic acid residues

Cited by 26 publications

References 20 publications

Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses

Racemisation and human cataract. d-Ser, d-Asp/Asn and d-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Contact Info

Product

Resources

About