The interplay between aromatic stacking and hydrogen bonding in nucleobases has been investigated via high-level quantum chemical calculations. The experimentally observed stacking arrangement between consecutive bases in DNA and RNA/DNA double helices is shown to enhance their hydrogen bonding ability as opposed to gas phase optimized complexes. This phenomenon results from more repulsive electrostatic interactions as is demonstrated in a model system of cytosine stacked offset-parallel with substituted benzenes. Therefore, the H-bonding capacity of the N3 and O2 atoms of cytosine increases linearly with the electrostatic repulsion between the stacked rings. The local hardness, a density functional theory-based reactivity descriptor, appears to be a key index associated with the molecular electrostatic potential (MEP) minima around H-bond accepting atoms, and is inversely proportional to the electrostatic interaction between stacked molecules. Finally, the MEP minima on surfaces around the bases in experimental structures of DNA and RNA–DNA double helices show that their hydrogen bonding capacity increases when taking more neighboring (intra-strand) stacking partners into account.

A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3–CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics.

The present work focuses on the influence of aromatic stacking on the ability of an aromatic nitrogen base to accept a hydrogen bond. Substituent effects were studied at the MP2 level for 10 complexes of a substituted benzene stacked with pyridine in a parallel offset conformation. The interaction energies between each substituted benzene and pyridine were analyzed in terms of Hartree-Fock, correlation, and electrostatic contributions. It appears that the basicity of pyridine is directly related to the electrostatic interaction between the cycles. It increases with increasing electron donating character of the benzene substituents. Also, density functional theory based descriptors such as global and local hardnesses and the benzene ring polarizability are found to adequately predict the interaction energy. These findings may be important in the study of DNA/ RNA chains.

General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. For example, hydrolysis/phosphorolysis of the N-glycosidic bond in nucleosides and nucleotides commonly involves the protonation of the leaving nucleobase concomitant with nucleophilic attack. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. This enzyme binds the purine base of the substrate between the aromatic side-chains of Trp83 and Trp260. Here, we show via quantum chemical calculations that face-to-face stacking can raise the pKa of a heterocyclic aromatic compound by several units. Site-directed mutagenesis combined with substrate engineering demonstrates that Trp260 catalyzes the cleavage of the glycosidic bond by promoting the protonation of the purine base at N-7, hence functioning as an alternative to general acid catalysis.

Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concentration dependence of the relative distribution of monomer HL and dimer (HL)(2) species, the apparent dissociation constant (K(D)) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding K(D) values quantified over a range of 10(-10)-10(-4) M. The critical importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4.

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Ribonucleases (RNases) catalyze the cleavage of the phosphodiester bond in RNA up to 10 15 -fold, as compared with the uncatalyzed reaction. High resolution crystal structures of these enzymes in complex with 3-mononucleotide substrates demonstrate the accommodation of the nucleophilic 2-OH group in a binding pocket comprising the catalytic base (glutamate or histidine) and a charged hydrogen bond donor (lysine or histidine). Ab initio quantum chemical calculations performed on such Michaelis complexes of the mammalian RNase A (EC 3.1.27.5) and the microbial RNase T 1 (EC 3.1.27.3) show negative charge build up on the 2-oxygen upon substrate binding. The increased nucleophilicity results from stronger hydrogen bonding to the catalytic base, which is mediated by a hydrogen bond from the charged donor. This hitherto unrecognized catalytic dyad in ribonucleases constitutes a general mechanism for nucleophile activation in both enzymic and RNAcatalyzed phosphoryl transfer reactions.RNases have been the subject of landmark research in areas ranging from basic protein chemistry to enzymology to protein folding and crystallography. These enzymes catalyze the intramolecular nucleophilic displacement of the 5Ј-leaving group by the attacking 2Ј-hydroxyl group in RNA, forming a 2Ј,3Ј-cyclophosphate (see Fig. 1). The nucleophilic attack occurs inline, in a postulated trigonal bipyramidal transition state, implying a catalytic base and acid on either side of the scissile bond (1, 2). X-ray crystallographical and site-directed mutagenesis experiments have provided substantial insight in the structure-function relationship of two unrelated families of ribonucleases. RNase A, (bovine pancreatic ribonuclease A) (EC 3.1.27.5) (3) and RNase T 1 , (EC 3.1.27.3) (4, 5) (and references therein) are the best characterized members of the mammalian and the microbial enzymes, respectively. The mammalian enzymes depend on two histidines for acid/base catalysis, whereas a histidine/glutamic acid pair is found in the microbial enzymes (6).The active site of RNase T 1 is composed of the side chains of Tyr-38, His-40, Glu-58, Arg-77, His-92, and Phe-100 (see Fig. 2a). With the exception of Arg-77, all these amino acids have been shown to take part in catalysis (5). Both the His-40 and Glu-58 side chains are in the direct vicinity of the 2Ј-nucleophile. pH dependence studies have shown Glu-58 to be unprotonated and His-40 to be protonated at the onset of catalysis, proving that Glu-58 is the catalytic base accepting a proton from the 2Ј-nucleophile (7). The protonated His-92 located at the opposite side of the active site functions as the catalytic acid. Removal of the His-40 side chain leads to a 6500-fold decrease in the second-order rate constant k cat /K m suggesting an important catalytic role for this residue in the wild type enzyme. His-40 is believed to polarize the 2Ј-hydroxyl group and to properly orientate Glu-58 toward the substrate (8, 9). However, the actual role of this residue is not totally clarified at present. Despite the l...

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