4968 Nucleic Acids Research, 2012, Vol. 40, No. 11 Figure 1. Size-exclusion chromatography shows formation of a transamidosome complex by ND-AspRS, GatCAB and tRNAAsn. (A) Comparison of isolated ND-AspRS and ND-AspRS/tRNAAsp and ND-AspRS/tRNAAsn complexes. The observed association enabled an estimation of the KD value for binding of each tRNA to ND-AspRS (see ‘Materials and Methods’ section). KD values for the binding to ND-AspRS are 7.9 and 21.4 mM for tRNAAsp and tRNAAsn, respectively. (B) Comparison between free GatCAB and GatCAB/ tRNAAsn complexes. The KD value for this association is 2.1 mM. (C) Elution proﬁle of a mixture containing GatCAB, ND-AspRS and tRNAAsn. (D) SDS–PAGE proﬁle of a SEC fraction (lane 3) compared to GatCAB alone (lane 1) and ND-AspRS alone (lane 2). (E) TLC plate demonstrating that the complex eluted in the ﬁrst peak (termed ‘isolated’) is able to produce Asn-tRNAAsn alone, or when supple- mented with excess tRNAAsn (termed ‘+2 mM tRNA’), compared to a negative control where GatCAB was omitted. All partners were mixed to a ﬁnal concentration of 20 mM each. All KD values determined varied within 10%. Figure 2. Characterization of the complexes involving Hp ND-AspRS, GatCAB, tRNAAsn and tRNAAsp by DLS and analysis of the stability of the transamidosome under catalytic and non-catalytic conditions. Each value is an average of ﬁve independent measurements with errors <5%. All the experiments were conducted under the same con- ditions as size chromatography experiments (see ‘Materials and Methods’ section). Ternary 1, 2 and 3 all refer to the transamidosome under non-catalytic (1), aspartylation (2) and aspartylation/ transamidation (3) conditions. diameter in the presence of small substrates, e.g. under catalytic conditions. The particle size decreased from 13.6 nm under non-catalytic conditions to 10.9 nm under aspartylation conditions (i.e. presence of Asp and ATP), and from 13.6 to 12.9 nm under transamidation conditions (i.e. presence of Asp, ATP and Gln). Furthermore, gel-ﬁltration with a mix of GatCAB, ND-AspRS and preformed Asp-tRNAAsn revealed elution of the binary GatCAB/Asp-tRNAAsn complex and a separate ND-AspRS peak (data not shown). These results suggest that charging of tRNAAsn may trigger destabilization of the transamidosome (Figure 2, lanes 7–9), apparently through release of ND-AspRS. Hence the Hp Asn- transamidosome displays completely different dynamics than does its T. thermophilus counterpart (21). Helicobacter pylori ND-AspRS, an enzyme with dual kinetic behavior Table 1 summarizes the steady-state kinetic parameters of Hp ND-AspRS for aminoacylation of tRNAAsp and