When planning a mutation to test some hypothesis, one crucial question is whether the new side chain is compatible with the existing structure; only if it is compatible can the interpretation of mutational results be straightforward. This paper presents a simple way of using the sensitive geometry of all-atom contacts (including hydrogens) to answer that question. The interactive MAGE/PROBE system lets the biologist explore conformational space for the mutant side chain, with an interactively updated kinemage display of its all-atom contacts to the original structure. The Autobondrot function in PROBE systematically explores that same conformational space, outputting contact scores at each point, which are then contoured and displayed. These procedures are applied here in two types of test cases, with known mutant structures. In ricin A chain, the ability of a neighboring glutamate to rescue activity of an active-site mutant is modeled successfully. In T4 lysozyme, six mutations to Leu are analyzed within the wild-type background structure, and their Autobondrot score maps correctly predict whether or not their surroundings must shift significantly in the actual mutant structures; interactive examination of contacts for the conformations involved explains which clashes are relieved by the motions. These programs are easy to use, are available free for UNIX or Microsoft Windows operating systems, and should be of significant help in choosing good mutation experiments or in understanding puzzling results.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic l-Asp/l-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of l-Asp by l-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (l-Asp/l-isoAsp)-X bond and of the X-(l-Asp/l-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either l-Asp or l-isoAsp, these identify one or the other form. Characterization and relative quantification of l-Asp/l-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1–7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41–46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.

Conserved deamidation of PKA catalytic subunit isozymes Cα and Cβ—more than 25% at Asn2 in vivo in both cases—has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7:457–469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the “repair” of isoAsp to Asp by the action of protein l-isoaspartyl (d-aspartyl) O-methyl transferase (PIMT). l-Succinimide is prone to racemization to d-succinimide, which may hydrolyze to d-isoAsp- and d-Asp-containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high-performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp- and isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of d-isoAsp- and d-Asp-containing T1 fragments in addition to the l-isomers. The small amount of the l-isoAsp isomer, representing only part of the d-isoAsp isomer, and the relatively large amounts of the l-Asp and d-Asp isomers argues for an effective action of PIMT present in cardiac tissue.

The PSI-BLAST algorithm has been acknowledged as one of the most powerful tools for detecting remote evolutionary relationships by sequence considerations only. This has been demonstrated by its ability to recognize remote structural homologues and by the greatest coverage it enables in annotation of a complete genome. Although recognizing the correct fold of a sequence is of major importance, the accuracy of the alignment is crucial for the success of modeling one sequence by the structure of its remote homologue. Here we assess the accuracy of PSI-BLAST alignments on a stringent database of 123 structurally similar, sequence-dissimilar pairs of proteins, by comparing them to the alignments defined on a structural basis. Each protein sequence is compared to a nonredundant database of the protein sequences by PSI-BLAST. Whenever a pair member detects its pair-mate, the positions that are aligned both in the sequential and structural alignments are determined, and the alignment sensitivity is expressed as the percentage of these positions out of the structural alignment. Fifty-two sequences detected their pair-mates (for 16 pairs the success was bi-directional when either pair member was used as a query). The average percentage of correctly aligned residues per structural alignment was 43.5 ± 2.2%. Other properties of the alignments were also examined, such as the sensitivity vs. specificity and the change in these parameters over consecutive iterations. Notably, there is an improvement in alignment sensitivity over consecutive iterations, reaching an average of 50.9 ± 2.5% within the five iterations tested in the current study.

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a β-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence–sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern {H-x(2)-[PTV]-x(4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC]-[GA]} for this family.