Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

doi: 10.1073/pnas.1120181109

Figure Lengend Snippet: Binding of NRne to anionic liposomes occurs through electrostatic interactions and affects the conformation of the protein. (A) NRne–liposome binding spin assays using different types of phospholipids. NRne (16 μM) was mixed with neutral, anionic, or cationic liposomes (1.35 mg/mL) as described in SI Materials and Methods. The total reaction mixture (T), supernatant (S), and pellet (P) were separated by 10% SDS/PAGE and analyzed by Coomassie Blue staining. (B) NRne interacts specifically with anionic liposomes through electrostatic interaction. Individual pellets (P), as shown in A, were divided and suspended in 100 mM sodium carbonate or 1 M NaCl, incubated for 30 min on ice, and repelleted using the procedure described in SI Materials and Methods. The repellet and supernatant are denoted P1 and S1, respectively. (C and D) Detection of conformational changes in NRne bound to the different types of liposomes by CD difference spectroscopy using a specifically designed tandem cuvettete (see Fig. S1A and Materials and Methods for details). The mixture conditions of protein and individual types of liposomes are identical to those used in the spin assays shown in A. CD difference spectra corresponding to individual types of NRne–liposome mixtures are shown. The affinity of NRne and liposomes was calculated by monitoring conformational changes upon binding as observed by CD difference spectroscopy (Materials and Methods). NRne (1 μM) was mixed with different concentrations of liposomes (0–20 μM). Data were fitted to a one-site binding hyperbolic curve by OriginPro software and are presented as mean values with SDs calculated from three independent experiments. NRne binds to the anionic liposomes with a high affinity (solid line, Kd = 298 ± 29 nM). NRne mixed with cationic liposomes shows no binding (dashed line).

Article Snippet: Data fitted to a one-site binding hyperbolic curve using OriginPro software (OriginLab) yielded an apparent dissociation constant ( K d ) of 298 ± 29 nM , demonstrating high-affinity binding of NRne to anionic liposomes.

Techniques: Binding Assay, SDS Page, Staining, Incubation, Spectroscopy, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

doi: 10.1073/pnas.1120181109

Figure Lengend Snippet: Binding to liposomes stabilizes the folding state of NRne and increases the enzymatic activity by increasing substrate affinity. (A and B) In vitro cleavage assays. As described in SI Materials and Methods, 20 pmol of BR13 or GGG-RNAI was incubated with 1 μg of liposome-free NRne (control) or liposome-bound (anionic liposomes) NRne at 37 °C. Aliquots taken after 2, 4, 6, 8, and 10 min (BR13) or 10, 20, 30, 40, and 50 min (GGG-RNAI) of incubation were separated on 20% or 8% sequencing gels, respectively. The position of bands corresponding to the substrates and products of cleavage are indicated at the left of each panel. (C) Michaelis–Menten analysis of BR13 cleavage by NRne in the presence (NRne+anionic liposomes) or absence (NRne) of anionic liposomes. The concentration of NRne was 2.5 nM in each sample; the concentration of substrate varied between 2.5 nM and 50 nM. The vo values reflecting the accumulation of cleavage product were fitted to a one-site binding hyperbolic curve using OriginPro software and are presented as mean values with SDs calculated from three independent experiments. (D) Thermally induced transition curves of NRne obtained by recording θ at 222 nm. The curves represented by filled and open circles correspond to the signal for NRne in the absence of anionic liposomes upon gradual temperature increase (from 10–60 °C, filled circles) or decrease (from 60–10 °C, open circles), respectively. The same measurements were performed for NRne in the presence of anionic liposomes (filled and open triangles, respectively). (E) Effect of temperature on CD spectra of NRne. Far UV CD spectra of NRne (amino acids 1–499) in the absence (line 1) and presence (line 3) of anionic liposomes as well as of anionic liposomes alone (line 5) were recorded at 25 °C. The samples were heated gradually to 60 °C and then were cooled; the CD spectra were recorded again at 25 °C (lines 2, 4, and 6, respectively). The compositions of the analyzed samples were the same as in the experiments presented in Fig. 2. (F) Thermal inactivation of NRne. Cleavage assays of BR13 were performed with NRne before (lane 1) and after (lane 2) thermal inactivation or with the liposome-bound variant before (lane 4) and after (lane 4) thermal inactivation using the NRne samples that were used for recording the CD spectra shown in D. The mixture conditions of protein and anionic liposomes are identical to those shown in A. Aliquots withdrawn after 4 min incubation were analyzed on a 20% sequencing gel.

Article Snippet: Data fitted to a one-site binding hyperbolic curve using OriginPro software (OriginLab) yielded an apparent dissociation constant ( K d ) of 298 ± 29 nM , demonstrating high-affinity binding of NRne to anionic liposomes.

Techniques: Binding Assay, Activity Assay, In Vitro, Incubation, Sequencing, Concentration Assay, Software, Variant Assay