Does deamidation cause protein unfolding? A top‐down tandem mass spectrometry study

Deamidation is a nonenzymatic post-translational modification of asparagine to aspartic acid or glutamine to glutamic acid, converting an uncharged amino acid to a negatively charged residue. It is plausible that deamidation of asparagine and glutamine residues would result in disruption of a proteins'' hydrogen bonding network and thus lead to protein unfolding. To test this hypothesis Calmodulin and B2M were deamidated and analyzed using tandem mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The gas phase hydrogen bonding networks of deamidated and nondeamidated protein isoforms were probed by varying the infra-red multi-photon dissociation laser power in a linear fashion and plotting the resulting electron capture dissociation fragment intensities as a melting curve at each amino acid residue. Analysis of the unfolding maps highlighted increased fragmentation at lower laser powers localized around heavily deamidated regions of the proteins. In addition fragment intensities were decreased across the rest of the proteins which we propose is because of the formation of salt-bridges strengthening the intramolecular interactions of the central regions. These results were supported by a computational flexibility analysis of the mutant and unmodified proteins, which would suggest that deamidation can affect the global structure of a protein via modification of the hydrogen bonding network near the deamidation site and that top down FTICR-MS is an appropriate technique for studying protein folding.     

Specific glutamine and asparagine residues of gamma-S crystallin are resistant to in vivo deamidation

It has been hypothesized that resistance to nonenzymatic deamidation of asparagine and glutamine residues may be an important determinant of protein stability in vivo. As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as gamma-S crystallin that were synthesized during the fetal-embryonic periods of development. Total protein from the fetal-embryonic region of old human lenses was digested with trypsin, followed by resolution of tryptic fragments containing amidated and deamidated forms using high pressure liquid chromatography-reverse phase chromatography together with synthetic peptide standards and mass spectral analysis. The results demonstrate no detectable deamidation of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from gamma-S crystallin from old human lenses, consistent with the hypothesis that very long-lived proteins can contain asparagine and glutamine residues that are extremely resistant to in vivo deamidation.     

Characterization of glutamine deamidation in a long, repetitive protein polymer via bioconjugate capillary electrophoresis

We describe a novel method for the determination of glutamine deamidation in a long protein polymer via bioconjugate capillary electrophoresis. Since the current best technique for detection of glutamine (or asparagine) deamidation is mass spectrometry, it is practically impossible to precisely detect the degree of deamidation (i.e., how many residues are deamidated in a polypeptide) in a large protein containing a significant number of glutamine (or asparagine) residues, because the mass difference between native and deamidated residues is just 1 atomic mass unit. However, by covalently attaching polydisperse protein polymers (337 residues) to a monodisperse DNA oligomer (22 bases), the degree of glutamine deamidation, which could not be determined accurately by mass spectrometry, was resolved by free-solution capillary electrophoresis. Electrophoretic separations were carried out after different durations of exposure of the protein to a cyanogen bromide cleavage reaction mixture, which is a general treatment for the purpose of removing an oligopeptide affinity purification tag from fusion proteins. For protein polymers with increasing extents of deamidation, the electromotive force of DNA + polypeptide conjugate molecules increases due to the introduced negative charge of deamidated glutamic acid residues, and consequently CE analysis reveals increasing differences in the electrophoretic mobilities of conjugate molecules, which qualitatively shows the degree of deamidation. Peak analysis of the electropherograms enables quantitative determination of the first four deamidations in a protein polymer. A first-order rate constant of 0.018 h(-1) was determined for the deamidation of a single glutamine residue in the protein polymer during the cyanogen bromide cleavage reaction.     

Asparagine deamidation and the role of higher order protein structure

The 'protein world' exhibits additional complexity caused by post-translational modifications. One such process is nonenzymic deamidation of asparagine which is controlled partly by primary sequence, but also higher order protein structure. We have studied the deamidation of an N-terminal peptide in muscle glyceraldehyde 3-phosphate dehydrogenase to relate three-dimensional structure, proteolysis, and deamidation. This work has significant consequences for identification of proteins using peptide mass fingerprinting.     

Prediction of Spontaneous Protein Deamidation from Sequence-Derived Secondary Structure and Intrinsic Disorder

Asparagine residues in proteins undergo spontaneous deamidation, a post-translational modification that may act as a molecular clock for the regulation of protein function and turnover. Asparagine deamidation is modulated by protein local sequence, secondary structure and hydrogen bonding. We present NGOME, an algorithm able to predict non-enzymatic deamidation of internal asparagine residues in proteins in the absence of structural data, using sequence-based predictions of secondary structure and intrinsic disorder. Compared to previous algorithms, NGOME does not require three-dimensional structures yet yields better predictions than available sequence-only methods. Four case studies of specific proteins show how NGOME may help the user identify deamidation-prone asparagine residues, often related to protein gain of function, protein degradation or protein misfolding in pathological processes. A fifth case study applies NGOME at a proteomic scale and unveils a correlation between asparagine deamidation and protein degradation in yeast. NGOME is freely available as a webserver at the National EMBnet node Argentina, URL: http://www.embnet.qb.fcen.uba.ar/ in the subpage “Protein and nucleic acid structure and sequence analysis”.     

Glutamine deamidation destabilizes human gammaD-crystallin and lowers the kinetic barrier to unfolding

Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human gammaD-crystallin (HgammaD-Crys) is a two-domain beta-sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of HgammaD-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 degrees C, or urea at pH 3.0, 20 degrees C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of HgammaD-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.     

Detection, evaluation and minimization of nonenzymatic deamidation in proteomic sample preparation

Identification of deamidated sites in proteins is commonly used for assignment of N-glycosylation sites. It is also important for assessing the role of deamidation in vivo. However, nonenzymatic deamidation occurs easily in peptides under conditions commonly used in treatment with trypsin and PNGase F. The impact on proteomic sample preparation has not yet been evaluated systematically. In addition, the (13)C peaks of amidated peptides can be misassigned as monoisotopic peaks of the corresponding deamidated ones in database searches. The 19.34 mDa mass difference between them is proposed as a means for eliminating the resulting false positive identifications in large-scale proteomic analysis. We evaluated five groups of proteomic data, obtained mainly through an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)-reverse phase (RP) chromatography sequence, and ascertained that nonenzymatic asparagine deamidation occurred to some extent on 4-9% of the peptides, resulting in the false positive identification of many N-glycosylation sites. A comprehensive investigation indicated that the chief causative factors were the mildly alkaline pH and prolonged incubations at 37 °C during proteomic sample preparation. An improved protocol is proposed featuring tryptic digestion at pH 6 and deglycosylation at pH 5, resulting in a significant decrease in nonenzymatic deamidation while conserving adequate digestion efficiency. The number of identified deamidation sites was improved significantly by increasing the sample loading amount in liquid chromatography-tandem MS. This permitted the identification of a significant number of glutamine deamidation sites, which featured sequence motifs largely different from those for asparagine deamidation: -Q-V-, -Q-L- and -Q-G- and, to a lesser extent, -Q-A- and -Q-E-.     

Structural Effects of Protein Aging: Terminal Marking by Deamidation in Human Triosephosphate Isomerase

Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.     

N-acyl substituted 7-amino-4-chloroisocoumarin: a peptide degradation model via an imide mechanism

During the coupling reaction between 3-alkoxy-7-amino-4-chloroisocoumarin and N-acyl alanine dipeptide, an unexpected deamidation reaction was observed. The proposed mechanism for this reaction involved the formation of an imide intermediate which after cleavage led to the release of amino acid moiety. The described deamidation reaction represents the first chemical model involving a non-peptidic moiety, which mimics biological and chemical deamidation processes occurring in proteins or peptides incorporating an asparagine or a glutamine residue.     

What a Difference a Dalton Makes: Bacterial Virulence Factors Modulate Eukaryotic Host Cell Signaling Systems via Deamidation

SUMMARY

Pathogenic bacteria commonly deploy enzymes to promote virulence. These enzymes can modulate the functions of host cell targets. While the actions of some enzymes can be very obvious (e.g., digesting plant cell walls), others have more subtle activities. Depending on the lifestyle of the bacteria, these subtle modifications can be crucially important for pathogenesis. In particular, if bacteria rely on a living host, subtle mechanisms to alter host cellular function are likely to dominate. Several bacterial virulence factors have evolved to use enzymatic deamidation as a subtle posttranslational mechanism to modify the functions of host protein targets. Deamidation is the irreversible conversion of the amino acids glutamine and asparagine to glutamic acid and aspartic acid, respectively. Interestingly, all currently characterized bacterial deamidases affect the function of the target protein by modifying a single glutamine residue in the sequence. Deamidation of target host proteins can disrupt host signaling and downstream processes by either activating or inactivating the target. Despite the subtlety of this modification, it has been shown to cause dramatic, context-dependent effects on host cells. Several crystal structures of bacterial deamidases have been solved. All are members of the papain-like superfamily and display a cysteine-based catalytic triad. However, these proteins form distinct structural subfamilies and feature combinations of modular domains of various functions. Based on the diverse pathogens that use deamidation as a mechanism to promote virulence and the recent identification of multiple deamidases, it is clear that this enzymatic activity is emerging as an important and widespread feature in bacterial pathogenesis.     

Modifications of therapeutic proteins: challenges and prospects

The production of therapeutic proteins is one of the fastest growing sectors of the pharmaceutical industry. However, most proteins used in drug therapy require complex post-translational modifications for efficient secretion, drug efficacy and stability. Common protein modifications include variable glycosylation, misfolding and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, and proteolysis. These modifications not only pose challenges for accurate and consistent bioprocessing, but also may have consequences for the patient in that incorrect modifications or aggregation may lead to an immune response to the protein therapeutic. This review provides examples of analytical and preventative advances that have been devised to meet these challenges, and insights into how further advances can improve the efficiency and safety in manufacturing recombinant proteins.     

Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry

Although deamidation at asparagine and glutamine has been found in numerous studies of a variety of proteins, in almost all cases the analytical methodology that was used could detect only a single site of deamidation. For the extensively studied case of reduced bovine ribonuclease A (13,689 Da), only Asn67 deamidation has been demonstrated previously, although one study found three monodeamidated fractions. Here top down tandem mass spectrometry shows that Asn67 deamidation is extensive before Asn71 and Asn94 react; these are more than half deamidated before Asn34 reacts, and its deamidation is extensive before that at Gln74 is initiated. Except for the initial Asn67 site, these large reactivity differences correlate poorly with neighboring amino acid identities and instead indicate residual conformational effects despite the strongly denaturing media that were used; deamidation at Asn67 could enhance that at Asn71, and these enhance that at Gln74. This success in the site-specific quantitation of deamidation in a 14 kDa protein mixture, despite the minimal 1 Da (-NH2 --> -OH) change in the molecular mass, is further evidence of the broad applicability of the top down MS/MS methodology for characterization of protein posttranslational modifications.     

Protein deamidase from germinating wheat grains.

A new enzyme catalyzing the deamidation of seed storage proteins was found in germinating wheat grains and was partially purified. It also acts on egg lysozyme, horse hemoglobin and reduced RNAse, glutamine and Gly-L-Gln-L-Tyr. No activity was observed when using ovalbumin, serum albumin, RNAse, insulin, asparagine and an asparagine-containing peptide. Only glutaminyl residues appear to be deamidated by this enzyme. It differs from transglutaminase and proved to be a true protein deamidase.     

Deamidation in human gamma S-crystallin from cataractous lenses is influenced by surface exposure

A major component of human nuclear cataracts is water-insoluble, high molecular weight protein. A significant component of this protein is disulfide bonded gamma S-crystallin that can be reduced to monomers by dithiothreitol. Analysis of this reduced gamma S-crystallin showed that deamidation of glutamine and asparagine residues is a principal modification. Deamidation is one of the modifications of lens crystallins associated with aging and cataractogenesis. One proposed hypothesis of cataractogenesis is that it develops in response to altered surface charges that cause conformational changes, which, in turn, permit formation of disulfide bonds and crystallin insolubility. This report, showing deamidation among the disulfide bonded gamma S-crystallins from cataractous lenses, supports this hypothesis.     

New genetic variants of parotid salivary amylase.

Isoelectric focusing can reveal at least two heterozygotes of the Amy1R class of variants. Family data on these variants show co-dominant inheritance for both alleles with respect to the normal Amy1A allele. The frequency of the alleles Amy1R1 and Amy1R2 in the Dutch population (n = 144) amounts to 0.006 and 0.03, respectively. Electrophoretic analysis of whole parotid saliva from different Amy1R phenotypes and of purified normal and Amy1R1 gene products indicates that the variant proteins differ from the normal protein by enhanced deamidation of asparagine and/or glutamine residues.     

Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A.

Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.     

Why does ribonuclease irreversibly inactivate at high temperatures?

The mechanism of irreversible thermoinactivation of bovine pancreatic ribonuclease A in the pH range relevant to enzymatic catalysis has been elucidated. At 90 degrees C and pH 4, the enzyme inactivation is caused by hydrolysis of peptide bonds at aspartic acid residues (the main process) and deamidation of asparagine and/or glutamine residues. At 90 degrees C and neutral pH (pH 6 and 8), the enzyme inactivation is caused by a combination of disulfide interchange (the main process), beta-elimination of cystine residues, and deamidation of asparagine and/or glutamine residues. These four processes appear to demarcate the upper limit of thermostability of enzymes.     

Immunochemical Studies on Arachis hypogaea Proteins With Particular Reference to the Reserve Proteins. II. Protein Modification During Germination

α-Conarachin and α-arachin are metabolized differently during germination. The amount of α-conarachin decreases without the appearance of antigenic intermediary compounds. α-Arachin retains its original antigenic structure and becomes more anodic during germination, but a number of intermediary proteins, differing widely in electrophoretic mobility, form up to 14 days germination. The electrophoretic shift of α-arachin, detectable after 1 day of germination, might be caused by progressive deamidation of glutamine and asparagine. Decreases in amide content of protein extracted after germination support the hypothesis and suggest that α-arachin provides the first source of ammonia to the new seedling.     

An improved trypsin digestion method minimizes digestion-induced modifications on proteins

Trypsin digestion can induce artificial modifications such as asparagine deamidation and N-terminal glutamine cyclization on proteins due to the temperature and the alkaline pH buffers used during digestion. The amount of these artificial modifications is directly proportional to the incubation time of protein samples in the reduction/alkylation buffer and, more important, in the digestion buffer where the peptides are completely solvent exposed. To minimize these artificial modifications, we focused on minimizing the trypsin digestion time by maximizing trypsin activity. Trypsin activity was optimized by the complete removal of guanidine, which is a known trypsin inhibitor, from the digestion buffer. As a result, near complete trypsin digestion was achieved on reduced and alkylated immunoglobulin gamma molecules in 30 min. The protein tryptic fragments and their modification products were analyzed and quantified by reversed-phase liquid chromatography/tandem mass spectrometry using an in-line LTQ Orbitrap mass spectrometer. The reduction and alkylation reaction time was also minimized by monitoring the completeness of the reaction using a high-resolution time-of-flight mass spectrometer. Using this 30-min in-solution trypsin digestion method, little protocol-induced deamidation or N-terminal glutamine cyclization product was observed and cleaner tryptic maps were obtained due to less trypsin self-digestion and fewer nonspecific cleavages. The throughput of trypsin digestion was also improved significantly compared with conventional trypsin digestion methods.     

Sequence and structure determinants of the nonenzymatic deamidation of asparagine and glutamine residues in proteins.

The rates of deamidation of Asn and Gln residues in peptides and proteins depend upon both the identity of other nearby amino acid residues, some of which can catalyze the deamidation reaction of the Asn and Gln side chains, and upon polypeptide conformation. Proximal amino acids can be contiguous in sequence or brought close to Asn or Gln side chains by higher order structure of the protein. Local polypeptide conformation can stabilize the oxyanion transition state of the deamidation reaction and also enable deamidation through the beta-aspartyl shift mechanism. In this paper, the environments of Asn and Gln residues in known protein structures are examined to determine the configuration and identity of groups which participate in deamidation reactions. Sequence information is also analyzed and shown to support evolutionary selection against the occurrence of certain potentially catalytic amino acids adjacent to Asn and Gln in proteins. This negative selection supports a functional role for deamidation in those non-mutant proteins in which it occurs.