Browsing by Subject "specificity"

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Binding Of Flexible And Constrained Ligands To The Grb2 Sh2 Domain: Structural Effects Of Ligand Preorganization
(2010-10) Clements, John H.; DeLorbe, John E.; Benfield, Aaron P.; Martin, Stephen F.; Clements, John H.; DeLorbe, John E.; Benfield, Aaron P.; Martin, Stephen F.
Structures of the Grb2 SH2 domain complexed with a series of pseudopeptides containing flexible (benzyl succinate) and constrained (aryl cyclopropanedicarboxylate) replacements of the phosphotyrosine (pY) residue in tripeptides derived from Ac-pYXN-NH(2) (where X = V, I, E and Q) were elucidated by X-ray crystallography. Complexes of flexible/constrained pairs having the same pY + 1 amino acid were analyzed in order to ascertain what structural differences might be attributed to constraining the phosphotyrosine replacement. In this context, a given structural dissimilarity between complexes was considered to be significant if it was greater than the corresponding difference in complexes coexisting within the same asymmetric unit. The backbone atoms of the domain generally adopt a similar conformation and orientation relative to the ligands in the complexes of each flexible/constrained pair, although there are some significant differences in the relative orientations of several loop regions, most notably in the BC loop that forms part of the binding pocket for the phosphate group in the tyrosine replacements. These variations are greater in the set of complexes of constrained ligands than in the set of complexes of flexible ligands. The constrained ligands make more direct polar contacts to the domain than their flexible counterparts, whereas the more flexible ligand of each pair makes more single-water-mediated contacts to the domain; there was no correlation between the total number of protein-ligand contacts and whether the phosphotyrosine replacement of the ligand was preorganized. The observed differences in hydrophobic interactions between the complexes of each flexible/constrained ligand pair were generally similar to those observed upon comparing such contacts in coexisting complexes. The average adjusted B factors of the backbone atoms of the domain and loop regions are significantly greater in the complexes of constrained ligands than in the complexes of the corresponding flexible ligands, suggesting greater thermal motion in the crystalline state in the former complexes. There was no apparent correlation between variations in crystal packing and observed structural differences or similarities in the complexes of flexible and constrained ligands, but the possibility that crystal packing might result in structural variations cannot be rigorously excluded. Overall, it appears that there are more variations in the three-dimensional structure of the protein and the ligand in complexes of the constrained ligands than in those of their more flexible counterparts.
The Impact of DNA-DNA Base Pairing on CRISPR-Cas12a Target Specificity
(2022-05) Uebele, Allison; Russell, Rick
CRISPR-Cas12a is a highly specific Cas endonuclease characterized by reversibility during R-loop formation that allows Cas12a to more effectively discriminate against mismatches compared to Cas9. However, the role of DNA-DNA base pairing in Cas12a’s target specificity is not fully understood. CRISPR-Cas12a target specificity occurs primarily when contacts are formed reversibly. We hypothesize that the ability of DNA to reform DNA-DNA base pairs at the expense of RNA-DNA base pairs increases reversibility and therefore specificity. In the present work, we eliminate DNA-DNA base pairs, predicting that R-loop formation would be faster and less specific. To test this, single DNA mismatches were generated throughout R-loop positions in both a traditional duplex target and a “bubble” target that lacks Watson-Crick base pairs in the R-loop region. Cis-cleavage and binding data indicate that R-loop formation occurs more rapidly in the absence of DNA-DNA base pairing compared to in the duplexed target. Results also indicate that at certain positions, mismatch specificity is decreased when DNA-DNA base pairs are absent from the target sequence. A model is suggested where target specificity is reduced in the absence of DNA-DNA base pairing throughout the entirety of R-loop positions.
An investigation of memory specificity and generalization in young children and adults
(2020) Hipskind, Elizabeth; Preston, Alison
Optimal behavior in familiar and novel contexts depends on retrieval and consideration of past experiences. In adults, hippocampus supports retrieval of prior memories based on partially overlapping cues (Mack & Preston, 2016). Given that the hippocampus develops through childhood and adolescence (Keresztes et al., 2017), in the present research we investigated developmental differences in flexible memory retrieval during new experiences. Four-year-olds (N=15) and adults (N=20) learned a series of common object-novel shape associations. Following learning, participants were cued with a shape and tasked with retrieving the target object associate. On half of the trials, participants were cued with an identical shape from learning. On the remaining trials, participants were cued with a similar but non-identical shape morph, enabling examination of whether participants can flexibly generalize across similar but non-identical experiences to retrieve related memories. Accuracy and response times were measured for adults, and accuracy was measured for children. Both adults and children demonstrated reliable retrieval when cued with similar yet non-identical shapes. Whereas adults showed slower and less accurate retrieval for the non-identical versus identical cues, children showed no differences in retrieval as a function of cue similarity. These findings have important implications for our understanding of how mnemonic specificity and generalization interact across development. In particular, our findings suggest that mnemonic generalization in early childhood is a consequence of less detailed memory representation. Conversely, the more mature form of generalization evidenced in adulthood is accomplished through dual processing of the commonalities and specific differences between similar yet non-identical experiences.