Dynamic characterization of HLA-B*44 Alleles: A comparative molecular dynamics simulation study

Human Leukocyte Antigens (HLA) are highly polymorphic proteins that play a key role in the immune system. HLA molecule is present on the cell membrane of antigen-presenting cells of the immune system and presents short peptides, originating from the proteins of invading pathogens or self-proteins, to the T-cell Receptor (TCR) molecule of the T-cells. In this study, peptide-binding characteristics of HLA-B*44:02, 44:03, 44:05 alleles bound to three nonameric peptides were studied using molecular dynamics simulations. Polymorphisms among these alleles (Asp116Tyr and Asp156Leu) result in major differences in the allele characteristics. While HLA-B*44:02 (Asp116, Asp156) and HLA-B*44:03 (Asp116, Leu156) depend on tapasin for efficient peptide loading, HLA-B*44:05 (Tyr116, Asp156) is tapasin independent. On the other hand, HLA-B*44:02 and HLA-B*44:03 mismatch is closely related to transplant rejection and acute-graft-versus-host disease. In order to understand the dynamic characteristics, the simulation trajectories were analyzed by applying Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) calculations and hydrogen bonding analysis. Binding dynamics of the three HLA-B*44 alleles and peptide sequences are comparatively discussed. In general, peptide binding stability is found to depend on the peptide rather than the allele type for HLA-B*44 alleles.     

Synthesis and biological evaluation of two chemically modified peptide epitopes for the class I MHC protein HLA-B*2705

The T-cell receptor of a CD8(+) T-cell recognises peptide epitopes bound by class I major histocompatibility complex (MHC) glycoproteins presented in a groove on their upper surface. Within the groove of the MHC molecule are 6 pockets, two of which mostly display a high degree of specificity for binding amino acids capable of making conserved and energetically favourable contacts with the MHC. One type of MHC molecule, HLA-B*2705, preferentially binds peptides containing an arginine at position 2. In an effort to increase the affinity of peptides for HLA-B*2705, potentially leading to better immune responses to such a peptide, we synthesised two modified epitopes where the amino acid at position 2 involved in anchoring the peptide to the class I molecule was replaced with the alpha-methylated beta,gamma-unsaturated arginine analogue 2-(S)-amino-5-guanidino-2-methyl-pent-3-enoic acid. The latter was prepared via a multi-step synthetic sequence, starting from alpha-methyl serine, and incorporated into dipeptides which were fragment-coupled to resin-bound heptameric peptides yielding the target nonameric sequences. Biological characterisation indicated that the modified peptides were poorer than the native peptides at stabilising empty class I MHC complexes, and cells sensitised with these peptides were not recognised as well by cognate CD8(+) T-cells, where available, compared to those sensitised with the native peptide. We suggest that the modifications made to the peptide have decreased its ability to bind to the peptide binding groove of HLA-B*2705 molecules which may explain the decrease in recognition by cytotoxic T-cells when compared to the native peptide.     

A computational docking study on the pH dependence of peptide binding to HLA-B27 sub-types differentially associated with ankylosing spondylitis

A single amino acid difference (Asp116His), having a key role in a pathogenesis pathway, distinguishes HLA-B*27:05 and HLA-B*27:09 sub-types as associated and non-associated with ankylosing spondylitis, respectively. In this study, molecular docking simulations were carried out with the aim of comprehending the differences in the binding behavior of both alleles at varying pH conditions. A library of modeled peptides was formed upon single point mutations aiming to address the effect of 20 naturally occurring amino acids at the binding core peptide positions. For both alleles, computational docking was applied using Autodock 4.2. Obtained free energies of binding (FEB) were compared within the peptide library and between the alleles at varying pH conditions. The amino acid preferences of each position were studied enlightening the role of each on binding. The preferred amino acids for each position of pVIPR were found to be harmonious with experimental studies. Our results indicate that, as the pH is lowered, the capacity of HLA-B*27:05 to bind peptides in the library is largely lost. Hydrogen bonding analysis suggests that the interaction between the main anchor positions of pVIPR and their respective binding pocket residues are affected from the pH the most, causing an overall shift in the FEB profiles.     

Computational analysis on the binding of epitope peptide to human leukocyte antigen class I molecule A*2402 subtype

Immunological response induced by small amino peptide has attracted much recent attention in the field of immunotherapy. Wilms' tumor (WT1) protein is one of the potent tumor antigens inducing immunological response in mouse and human, because WT1 is over expressed in many types of leukemia and various kinds of solid tumors. A 9-mer peptide encoded in WT1 protein (CMTWNQMNL; amino acid 235-243) is known to serve as antigenic peptide for human leukocyte antigen (HLA)-A*2402 molecule. It was reported that the replacement of the second amino residue, which is deeply responsible for the peptide binding to HLA, induced strong immunological response compared to the natural peptide. In this study, 19 kinds of single amino substitutions were introduced at position 2 of this 9-mer WT1 peptide. We performed molecular dynamics simulation on the complex of each of WT1 epitope peptides and HLA-β2 micro globulin (β2m) molecule, and subsequently estimated the binding affinity using molecular mechanics/generalized-Born surface area method combined with normal mode analysis. Our computation indicated that the peptide containing M2Y or M2W mutation showed high binding affinity to the HLA-β2m molecule as well as the natural peptide. We have also examined the role of the residue at position 2 in peptide binding to HLA-β2m. The calculation showed that van der Waals interaction between the side chain of the residue at position 2 and hydrophobic residues inside B-pocket of HLA are important. These findings will be helpful to search other potent peptides that will enhance strong immunological response specific to HLA-A*2402 molecule.     

Computational investigation of peptide binding stabilities of HLA-B*27 and HLA-B*44 alleles

Major Histocompatibility Complex (MHC) is a cell surface glycoprotein that binds to foreign antigens and presents them to T lymphocyte cells on the surface of Antigen Presenting Cells (APCs) for appropriate immune recognition. Recently, studies focusing on peptide-based vaccine design have allowed a better understanding of peptide immunogenicity mechanisms, which is defined as the ability of a peptide to stimulate CTL-mediated immune response. Peptide immunogenicity is also known to be related to the stability of peptide-loaded MHC (pMHC) complex. In this study, ENCoM server was used for structure-based estimation of the impact of single point mutations on pMHC complex stabilities. For this purpose, two human MHC molecules from the HLA-B*27 group (HLA-B*27:05 and HLA-B*27:09) in complex with four different peptides (GRFAAAIAK, RRKWRRWHL, RRRWRRLTV and IRAAPPPLF) and three HLA-B*44 molecules (HLA-B*44:02, HLA-B*44:03 and HLA-B*44:05) in complex with two different peptides (EEYLQAFTY and EEYLKAWTF) were analyzed. We found that the stability of pMHC complexes is dependent on both peptide sequence and MHC allele. Furthermore, we demonstrate that allele-specific peptide-binding preferences can be accurately revealed using structure-based computational methods predicting the effect of mutations on protein stability.     

Molecular mechanism of the susceptibility difference between HLA-B*27:02/04/05 and HLA-B*27:06/09 to ankylosing spondylitis: substitution analysis,MD simulation,QSAR modelling,and in vitro assay

The human leukocyte antigen HLA-B27 is directly involved in the disease pathogenesis of ankylosing spondylitis (AS). HLA-B27 has a high degree of genetic polymorphism, with 105 currently known subtypes; the presence of aspartic acid at residue 116 (Asp116) has been found to play an essential role in AS susceptibility. Here, we systematically investigated the molecular mechanism of the susceptibility difference between the AS-associated subtypes HLA-B*27:02/04/05 and AS-unassociated subtypes HLA-B*27:06/09 to AS at sequence, structure, energetic and dynamic levels. In total seven variable residues were identified among the five studied HLA-B27 subtypes, in which Asp116 can be largely stabilized by a spatially vicinal, positively charged His114 through a salt bridge, while five other variable residues seem to have only a marginal effect on AS susceptibility. We also employed a quantitative structure–activity relationship approach to model the statistical correlation between peptide structure and affinity to HLA-B*27:05, a genetic ancestor of all other HLA-B27 subtypes and associated strongly with AS. The built regression predictor was verified rigorously through both internal cross-validation and external blind validation, and was then employed to identify potential HLA-B*27:05 binders from >20,000 cartilage-derived self-peptides. Subsequently, the binding potency of the top five antigenic peptides to HLA-B*27:05 was assayed in vitro using a FACS-based MHC stabilization experiment. Consequently, two (QRVGSDEFK and LRGAGTNEK) out of the five peptides were determined to have high affinity (BL₅₀ = 5.5 and 15.8 nM, respectively) and, as expected, both of them possess positively charged Lys at the C-terminus.     

Effect of chain length on the conformation and T cell recognition of synthetic hemagglutinin fragments

Circular dichroism and Fourier-transform infrared spectroscopies were used to compare the conformational mobility of 13-mer peptides covering the 317-329 region of the envelope protein hemagglutinin of human influenza A virus subtypes H1, H2 and H3 with that of their truncated deca- and nonapeptide analogs. These peptides were demonstrated to bind to the murine I-Ed major histocompatibility complex encoded class II and human HLA-B*2705 class I molecules. Despite the amino acid substitutions in the three 13-mer subtype sequences, no significant differences in the conformational properties could be shown. Deletion of the N-terminal three residues resulted in a shift to an increased alpha-helical conformer population in the 317-329 H1 peptide and the breakage of the 3(10) or weakly H-bonded (nascent) alpha-helix in the H2 and H3 peptides. The conformational change observed upon deletion did not influence the efficiency of I-Ed peptide interaction, however, the C-terminal Arg had a beneficial effect both on MHC class II and class I binding without causing any remarkable change in solution conformation.     

Evaluation of potential MHC-I allele-specific epitopes in Zika virus proteins and the effects of mutations on peptide-MHC-I interaction studied using in silico approaches

Zika virus (ZIKV) infection is a global health concern due to its association with microcephaly and neurological complications. The development of a T-cell vaccine is important to combat this disease. In this study, we propose ZIKV major histocompatibility complex I (MHC-I) epitopes based on in silico screening consensus followed by molecular docking, PRODIGY, and molecular dynamics (MD) simulation analyses. The effects of the reported mutations on peptide-MHC-I (pMHC-I) complexes were also evaluated. In general, our data indicate an allele-specific peptide-binding human leukocyte antigen (HLA) and potential epitopes. For HLA-B44, we showed that the absence of acidic residue Glu at P2, due to the loss of the electrostatic interaction with Lys45, has a negative impact on the pMHC-I complex stability and explains the low free energy estimated for the immunodominant peptide E-4 (IGVSNRDFV). Our MD data also suggest the deleterious effects of acidic residue Asp at P1 on the pMHC-I stability of HLA-B8 due to destabilization of the α-helix and β-strand. Free energy estimation further indicated that the mutation from Val to Ala at P9 of peptide E-247 (DAHAKRQTV), which was found exclusively in microcephaly samples, did not reduce HLA-B8 affinity. In contrast, the mutation from Thr to Pro at P2 of the peptide NS5−832 (VTKWTDIPY) decreased the interaction energy, number of intermolecular interactions, and adversely affected its binding mode with HLA-A1. Overall, our findings are important with regard to the design of T-cell peptide vaccines and for understanding how ZIKV escapes recognition by CD8 + T-cells.     

Medium-high resolution electrochemical genotyping of HLA-DQ2/DQ8 for detection of predisposition to coeliac disease

Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained.

Actinic Prurigo in Singaporean Chinese: A Positive Association with HLA‐DRB1*03:01

Studies have reported the association of human leukocyte antigen (HLA) genes with susceptibility to develop actinic prurigo (AP) in Caucasians, but there were no studies in Asian populations, including the Chinese. Our study was performed to determine if AP is associated with susceptibility or protective HLA alleles or haplotypes in Singaporean Chinese. All Chinese patients diagnosed with AP at National Skin Center, Singapore, from January 2002 to April 2015 were invited to participate in the study. Clinical data and phototesting results were collated, and HLA typing was performed. Among 14 patients included, 11 were male and the mean age was 49.6 (37.9–61.3) years. All patients did not have a family history of AP and none had mucosal involvement, as such these clinical features differed from Caucasian AP patients. The frequency of DRB1*03:01 in AP patients was significantly higher compared to healthy controls (43% vs 16%, P = 0.022, odds ratio (OR) 3.89). Concurrently, the frequency of HLA‐B*58:01‐DRB1*03:01 haplotype was also significantly increased (25% vs 7%, P = 0.004, OR 4.23). In conclusion, HLA‐DRB1*03:01 was associated with AP in Singaporean Chinese patients. This novel allelic association may possibly be utilized as a biological marker to aid in the diagnosis of AP in Chinese patients.     

An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants K_D towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a K_D of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

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Efficient Covalent Bond Formation in Gas-Phase Peptide–Peptide Ion Complexes with the Photoleucine Stapler

Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* _n L _m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N₂ from the L* diazirine rings underwent insertion into X?H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific ¹³C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H)⁺ ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H]⁺ ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide–peptide ion interactions in the gas phase.

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Pharmacomodulation of a ligand targeting the HBV capsid hydrophobic pocket

Hepatitis B virus (HBV) is a small enveloped retrotranscribing DNA virus and an important human pathogen. Its capsid-forming core protein (Cp) features a hydrophobic pocket proposed to be central notably in capsid envelopment. Indeed, mutations in and around this pocket can profoundly modulate, and even abolish, secretion of enveloped virions. We have recently shown that Triton X-100, a detergent used during Cp purification, binds to the hydrophobic pocket with micromolar affinity. We here performed pharmacomodulation of pocket binders through systematic modifications of the three distinct chemical moieties composing the Triton X-100 molecule. Using NMR and ITC, we found that the flat aromatic moiety is essential for binding, while the number of atoms of the aliphatic chain modulates binding affinity. The hydrophilic tail, in contrast, is highly tolerant to changes in both length and type. Our data provide essential information for designing a new class of HBV antivirals targeting capsid–envelope interactions.

Small-molecule binding to the Hepatitis B virus core protein hydrophobic pocket, a possible strategy for targeting viral particle assembly.     

Antibody-free lanthanide-based fluorescent probe for determination of protein tyrosine kinase and phosphatase activities

A single-labeled peptide probe for measuring peptide phosphorylation status was developed by using a phosphate sensitive terbium chelate. The activity of Abl protein tyrosine kinase and T-cell protein Tyrosine phosphatase (TC PTP) was monitored in real time. To study the probe design in detail, variable substrate peptide sequences, where the enzyme target site was located from two to five amino acids apart from the nearest tyrosine residue, were synthesized. The maximum change observed in fluorescence intensity after phosphorylation was up to 320%, when the phosphorylated tyrosine was located two amino acids from the lysine coupled to the phosphate sensitive terbium chelate, demonstrating an excellent performance for a homogeneous assay. Also the longer distance of five amino acids between the phosphorylated tyrosine residue and terbium chelate resulted up to 260% change in fluorescence intensity.

Electron Transfer Dissociation of Photolabeled Peptides. Backbone Cleavages Compete with Diazirine Ring Rearrangements

Gas-phase conformations and electron transfer dissociations of pentapeptide ions containing the photo-Leu residue (L*) were studied. Exhaustive conformational search including molecular dynamics force-field, semi-empirical, ab initio, and density functional theory calculations established that the photo-Leu residue did not alter the gas-phase conformations of (GL*GGK? + ?2H)²⁺ and (GL*GGK-NH₂?+?H)⁺ ions, which showed the same conformer energy ranking as the unmodified Leu-containing ions. This finding is significant in that it simplifies conformational analysis of photo-labeled peptide ions. Electron transfer dissociation mass spectra of (GL*GGK? + ?2H)²⁺, (GL*GGK-NH₂?+?2H)²⁺,(GL*GGKK?+?2H)²⁺, (GL*GLK?+?2H)²⁺, and (GL*LGK?+?2H)²⁺ showed 16 %–21 % fragment ions originating by radical rearrangements and cleavages in the diazirine ring. These side-chain dissociations resulted in eliminations of N₂H₃, N₂H₄, [N₂H₅], and [NH₄O] neutral fragments and were particularly abundant in long-lived charge-reduced cation-radicals. Deuterium labeling established that the neutral hydrazine molecules mainly contained two exchangeable and two nonexchangeable hydrogen atoms from the peptide and underwent further H/D exchange in an ion–molecule complex. Electron structure calculations on the charge-reduced ions indicated that the unpaired electron was delocalized between the diazirine and amide π* electronic systems in the low electronic states of the cation-radicals. The diazirine moiety in GL*GGK-NH₂was calculated to have an intrinsic electron affinity of 1.5 eV, which was further increased by the Coulomb effect of the peptide positive charge. Mechanisms are proposed for the unusual elimination of hydrazine from the photo-labeled peptide ions.

Synthesis of two peptides of α-bungarotoxin and the participation of the amino acid residue Trp-28 of the neurotoxin in the antigenicity of the molecule

Using the method of solid-phase peptide synthesis, two peptides have been synthesized, one of which corresponds to the central ring structure of -bungarotoxin (-BTX), while the second has in position 28 a Gly residue in place of the Trp in the first peptide, and their interrelationship with antitoxin antibodies has been investigated. It has been shown that the amino acid residue Trp-28 of -BTX, which is the contact residue in binding with the acetylcholine receptor, also participates directly in binding with the active centers of antibodies to -BTX.Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 628–631, July–August, 1997.     

Genetic polymorphisms of the A and B subunits of human coagulation factor XIII in mainland Italy and Sardinia: description of a new FXIIIA variant allele.

The distribution of the two alleles of FXIIIA and the three alleles of FXIIIB were studied in populations from mainland Italy and from Sardinia. The frequencies of the FXIIIA*2 allele were 0.266 and 0.265. The frequencies of FXIIIB*1 were 0.787 and 0.765; of B*2, 0.070 and 0.094; of B*3, 0.143 and 0.141. A new cathodal FXIIIA allele (A*7) was described in the Rome sample. No significant difference in the distribution of allele frequencies for either system was found between the two populations studied. For typing both markers, good results were also obtained by using whole blood instead of plasma.     

Molecular determinants in the bioactivation of the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Summary Nineteen analogs of the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have been used as probes to study the structural parameters that influence MAO-catalyzed oxidation. In this study, the efficiency of enzyme-catalyzed substrate oxidation was found to be unrelated to parameters such as the ionization potential, dipole moment, net atomic charge at C5 and the dihedral angle between the phenyl ring and the tetrahydropyridine moiety. Conformational analysis revealed that substitution at the C2 position of MPTP yields atropisomers. It is suggested that one of these atropisomers would be either inactive or substantially less active than the other. Therefore, the relative oxidative efficiency and toxicity of these compounds reported earlier may have been significantly underestimated. Based on the conformational analysis and other data, a rudimentary model of the MAO substrate site has been developed which partially explains the substrate specificities of MAO A and MAO B.Each substrate binding site can be divided into two regions, (a) an amine-binding pocket (for the tetrahydropyridine moiety), and (b) a bulky substituent region (for the phenyl group and its substituents). The length of the substrate binding site (measured along the long axis of MPTP) is approximately 8.5 Å, and the width of the amine-binding pocket is approximately 2.5 Å (from C3 to C5). The bulky substituent region contains a central area for binding the phenyl group of MPTP. This central area is flanked by two hydrophobic pockets, P2 and P3. In MAO A, the pocket P2-A is oriented 45–135° relative to the plane of the tetrahydropyridine moiety, with a radius of 3.1 Å from C2 of the phenyl ring. The radius of a similar but smaller pocket, P2-B, in MAO B, is approximately 2.7 Å. In MAO B, the pocket P3-B (radius 2.36 Å from C3) is larger than a similar pocket P3-A (radius 1.70 Å from C3) in MAO A. The foregoing characterization suggests that differences in the size and topography of both of the substituent pockets play an important role in determining the substrate specificities of these two isozymes.     

Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-acceptor specificity of the wild-type enzyme

The direct substrates of one protein kinase in a cell can be identified by mutation of the ATP binding pocket to allow an unnatural ATP analog to be accepted exclusively by the engineered kinase. Here, we present structural and functional assessment of peptide specificity of mutant protein kinases with unnatural ATP analogs. The crystal structure (2.8 A resolution) of c-Src (T338G) with N(6)-(benzyl) ADP bound shows that the creation of a unique nucleotide binding pocket does not alter the phospho-acceptor binding site of the kinase. A panel of optimal peptide substrates of defined sequence, as well as a degenerate peptide library, was utilized to assess the phospho-acceptor specificity of the engineered "traceable" kinases. The specificity profiles for the mutant kinases were found to be identical to those of their wild-type counterparts.     

Collision-Induced Dissociation of Diazirine-Labeled Peptide Ions. Evidence for Brønsted-Acid Assisted Elimination of Nitrogen

Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N₂ elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N₂ elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N₂ from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N₂ loss is proposed as a thermometer dissociation for peptide ion dissociations.