Concentration-dependence of the folding kinetics of OmpA into A8-35

conductance large 250-350 pS (state 2) after refolding of OmpA into A8-35. E Histogram of the probability of conductance after adding A8-35/OmpA complexes to the cis-compartment (upper panel) and the same amount of A8-35 (but without OmpA) to the trans-compartment of the Teflon chamber (lower panel).

We were interested, whether A8-35 would provide a similar protection to OmpA after incubation with the unfolded forms. The migration of refolded OmpA after trypsin digestion is shown in Figure 3.1C for OmpA refolded the LDAO (lane 6) and for OmpA refolded in A8-35 (lane 8). A8-35 protected the transmembrane domain of OmpA against proteolysis with trypsin, leading to the 24 kDa fragment that was observed previously for the proteolysis of lipid bilayer inserted OmpA (Surrey and Jähnig 1992).

Functional activity of OmpA was confirmed with single channel recordings (Figure 3.1D and 3.1E). For these experiments, the A8-35 refolded OmpA was reconstituted into black lipid films of diphytanoyl phosphatidylcholine (diphPC). Confirmation of A8-35 induced folding and reactivation is possible, because OmpA cannot be directly refolded into bilayers of diphPC (Figure 3.1D, trace 1). After reconstitution, OmpA formed small 4-16 pS channels (Figure 3.1D, state 1) as well as channels of larger, 250-320 pS conductance (Figure 3.1D, state 2). The small channel openings of detergent refolded OmpA had a conductance of 50 pS (Arora et al. 2000), larger than the conductance that we observed for OmpA reconstituted from OmpA/A8-35 complexes. However, we were able to obtain the same conductance for A8-35 refolded OmpA after reconstitution into diphPC bilayers, when we added the same concentration of A8-35 (but without OmpA) to the trans-compartment of the Teflon chamber prior to performing single channel recordings (Figure 3.1E, lower panel). We concluded that the initially observed reduced conductance was due to the sole presence of A8-35 in the cis-compartment, but not caused by alterations of the OmpA channel structure.

3.4.2 Concentration-dependence of the folding kinetics of OmpA into A8-35

To investigate the stoichiometry of OmpA interactions with A8-35 and the basic mechanism of OmpA folding into A8-35, we performed kinetic studies as a function of the concentration of amphipol A8-35 (Figure 3.2). In a set of different experiments, OmpA was reacted with A8-35 at selected ratios of 0.5, 1, 2, 6, 8, 10, 16 mg/mg. Since synthesis

of A8-35 polymers results in different degrees of polymerization with an average Mr of 8 kDa, this corresponds to averaged molar A8-35/OmpA ratios of 2.2, 4.4, 8.8, 26.3, 35, 43.8 and 70. The time courses of folding were determined by SDS-PAGE as described in Materials and Methods.

Figure 3.2 Kinetics of insertion and folding of OmpA into amphipol A8-35 determined at different concentrations of A8-35. A Each SDS-Polyacrylamide gels shows the formation of increasing amounts of the folded OmpA with increased incubation time after initiation of folding by denaurant-dilution of unfolded OmpA in urea with A8-35 solutions. Unfolded OmpA (U) migrated at 35 kDa, folded OmpA at 30 kDa (F). The OmpA concentration was 7.2 μM in all gels and the molar ratios amphipol/OmpA were 2.2, 4.4, 8.8, 26.3, 35, 43.8, and 70 (from the gel on the bottom to the gel on the top of panel A). B The fraction of folded OmpA was determined from the gels shown in panel A by densitometry and plotted as a function of time. All folding reactions were performed at 40°C in 10 mM Borax; 2 mM EDTA; pH 10.0.

Figure 3.2A shows the SDS-polyacrylamide gels obtained at the selected A8-35/OmpA ratios. Even at relatively low ratios, high refolding yields of OmpA were obtained within one hour. For analysis, the fraction of folded OmpA migrating at 30 kDa was determined relative to the total amount of OmpA migrating at 30 (F) and at 35 kDa (U) by densitometric analysis of the bands in each lane and then plotted as a function of time (Figure 3.2B) for each of the selected A8-35/OmpA ratios.

The time course of OmpA folding could not be well fitted by simple single-step kinetics, but in electrophoretic mobility analyses on the folding kinetics of OmpA, we observed only two bands at 35 and at 30 kDa representing the unfolded and folded forms

of OmpA, respectively. Intermediate forms of OmpA upon folding into A8-35 were not detected by SDS-PAGE. Since folding was measured by the relative increase of the 30 kDa form as a fraction of the total OmpA present and clearly a fast and a slow phase of formation of the 30 kDa form were observed (Figure 3.2B), our data indicate the presence of parallel pathways of OmpA folding into A8-35. To analyze the time course of OmpA folding into A8-35, we performed fits to three different kinetic models, assuming two parallel single folding steps. In these fits, we either assumed two parallel first-order steps, two parallel second-order steps or a first-order step parallel to a second-order step. A single-step second-order folding phase was observed previously for the folding of OmpA into lipid vesicles (Kleinschmidt and Tamm 2002) and we expected that it also would be observed in the present experiments, because the rates of interaction of OmpA with A8-35 should be faster with increasing A8-35 concentration.

The two rate constants, the relative contributions of the faster kinetic phase, and the fractions of OmpA folded at the end of the reaction were the fit parameters in all models and are shown in Table 3.1. In all 3 models, the relative contribution of the faster phase was independent of the concentration of A8-35 and also did not depend on the order of the fast and slow folding steps. On average, the faster process contributed 55 ± 9% to OmpA folding.

When two parallel first-order phases were fitted to the data, the rate constants k1

and k2 of the two folding processes did not depend on the A8-35 concentration. In contrast, the fits to the two models containing second-order folding steps indicated that the second order rate constants always decreased with increasing A8-35 concentration. This was unexpected and indicated that both folding pathways observed here must be of first order, since rate constants should be concentration independent. We therefore concluded that both the fast and the slow folding processes are of first order. The average first-order rate constants were 0.126 ± 0.020 min^–1 for the fast process and 0.0100 ± 0.0026 for the slow process, corresponding two half-times, τ =1/ 2 ln 2 / k

( )

Table 3.1 Rate constants of OmpA folding into A8-35 at different concentrations

A. Fits to kinetics composed of two first-order phases

T^a [P] ^b [A] ^c [A]/[P] k₁^d k₂^e f ^f R^g B. Fits to kinetics composed of two second-order phases

T^a [P] ^b [A] ^c [A]/[P] k₁^d k₂^e f ^f R^g C. Fits to kinetics composed of a second and a first order phase

T^a [P] ^b [A] ^c [A]/[P] k1d k2e f ^f R^g Fits of the data shown in Figure 3.2B were performed to three different models, composed of either two parallel first-order phases (A), two parallel second-order phases (B) or two parallel phases of first and of second order (C). The rate constants of the two kinetic phases, the relative contribution of the fast phase, and the final folding yield were free fit parameters.

atemperature, ^bconcentration of OmpA, ^cconcentration of amphipol A8-35, ^drate constant of the fast phase. ^erate constant of the slow phase, ^frelative contribution of the fast phase,

gfinal fractionof folded OmpA