Figure 1.

Standard protocol used for microscale preparation of NMR samples of OmpX reconstituted with the detergent Fos-10. In the first step, urea-unfolded OmpX is reconstituted in Fos-10 micelles. In the second step the volume of the solution of the mixed micelles in the refolding buffer is reduced to 50 μl, using centrifugal concentration devices with membranes of MWCO = 10 kDa. In each of four subsequent steps the concentrated protein solution is diluted with a three-fold excess of NMR buffer, and with the aforementioned centrifugal device the sample volume is again reduced to 50 μl. The protein and detergent concentrations, the MWCO of the centrifugal device, the reaction temperature, the reaction times and the repeats of individual steps indicated in the figure are the result of a procedure optimization described in the first part of this paper (the buffers are described in the Materials and Methods section). They are different from the conditions used previously with the same overall set-up⁹. The optimization was pursued by monitoring the detergent and protein concentrations in the solutions I–V. The solution V corresponds to the final sample, for which a 2D [¹⁵N,¹H]-correlation NMR experiment was also recorded (see Materials and Methods).