To date, the high resolution structure of only the C-terminal domain of TolA III has been resolved by X-ray crystallography (Lubkowski et al., 1999). TolA-I is the transmembrane domain which is not included in soluble constructs. TolA II is predicted to consist of long helices and, due to its probable flexibility, solution scattering is the best approach to shed light on the structure of TolA II-III. The solution structure of truncated TolA42-421 including central (domain II) and C-
terminal domains (domain III) but missing the N-terminal membrane spanning domain (domain I) was examined by SAXS. The schematic representation of TolA domains and localisation of TolA protein in E. coli is in Figure 3.14.
Figure 3.14 The structure of TolA from E. coli. a) Schematic of TolA domains b) Periplasmic localisation of TolA proteins. TolA I (cyan) is a transmembrane helix located on the inner membrane. TolA II (green) modeled by I-TASSER (Roy et al., 2010) is assumed to be a long -helical structure that spans the periplasmic space. TolA III (red) (PDB code: 3QDP) has a globular structure. Firstly, in order to ensure the homogeneity of TolA II-III, the concentration series of proteins was assessed by AUC. The TolA II-III samples were prepared at various concentrations in 50 mM sodium phosphate buffer, pH 7.4, 300 mM NaCl. According to Figure 3.15, The c(s) distribution of TolA II-III showed a single dominant peak with a sedimentation coefficient (s ) of 1.534 S in TolA
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II-III at all concentrations measured. This means that TolA II-III was present as a single species in solution. The low s value obtained from the experiment gave a clear indication of an elongated structure. The (estimated) s value of globular proteins can be estimated by their molecular weight. The lower experimental s value when comparing to theoretical s value indicates an extended non-globular structure (Lebowitz et al., 2002). The molecular mass of TolA II-III computed from sedimentation velocity experiment is 38.01 kDa which is slightly lower than the theoretical molecular mass of TolA II-III (39.66 kDa). As the AUC data confirmed TolA II-III is homogenous in solution, the TolA II-III sample is suitable for use in solution scattering experiments.
Figure 3.15 Sedimentation velocity of TolA II-III. a) Sedimentation velocity profiles. b) Sedimentation coefficient distributions c(s) at different concentrations. The TolA samples were in 50 mM sodium phosphate, pH 7.4, 300 mM NaCl.
SAXS curve of TolA at 5.3 mg/ml was recorded on the BM29 beamline at ESRF, Grenoble, France (Figure 3.16a). The P(r) showed a sign of an extended tail at the long distance and yielded a Dmax of 410 Å and a Rg of 118.7 ± 2.09 Å
(Figure 3.16b) whereas a Rg of 45.18 ± 3.70 Å was estimated by the Guinier
analysis. The disagreement of Rg between these two analyses indicated TolA II-
III possesses an elongated and flexible structure. The Guinier plot appeared to be a straight line which confirmed the homogeneity of TolA II-III in solution (Figure 3.16c). The Kratky plot of TolA lacks a bell-shaped peak when comparing to that of BSA (Figure 3.16d). Having a plateau in Kratky plot also supported that the TolA II-III structure is flexible and elongated.
Figure 3.16 SAXS data for TolA II-III. a) SAXS data (symbols) and fitting (line) by GNOM b) Distance distribution functions, P(r), calculated using GNOM c) Guinier analysis d) Kratky plot for TolA II-III compared to BSA. TolA II-III at 5.3 mg/ml in 50 mM sodium phosphate buffer, pH 7.4, 300 mM NaCl and BSA at 2.0 mg/ml in water were used in the measurement.
To further confirm the flexibility of TolA II-III, the SAXS curve was investigated using CRYSOL software (Svergun et al., 1995). CRYSOL is used to compute the theoretical scattering amplitude from atomic structure and compares the theoretical scattering curve to the experimental one. The TolA II structures
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modeled by I-TASSER (Roy et al., 2010) were then joined to the crystal structure of TolA III (PDB code: 3QDP) (Figure 3.17d). Three models at different lengths were made and compared to the experimental SAXS curve by CRYSOL (Figure 3.17a, b and c). The best fit was from model 2 which has a maximum length of 190 Å. However, none of these models were a perfect fit to the experimental data because the goodness of fit () was too large (it should be less than 1.1). Therefore, only one conformation is unlikely to truly represent the structure of TolA II-III. It is concluded that TolA II-III is likely to have more than one conformation in solution.
Figure 3.17 Theoretical SAXS profiles of TolA II-III models generated from CRYSOL fitted against the experimental SAXS profiles. Lines and symbols represent CRYSOL fitting and SAXS data, respectively a) Model 1. b) Model 2 and c) Model 3. d) TolA II-III structures modeled by I-TASSER (Roy et al., 2010) and optimised by Coot.
Consequently, to deal with this flexible system, EOM analysis of TolA II-III was carried out. The pool of TolA II-III models based on a sequence of TolA II and the crystal structure of TolA III was randomly created. 295 residues of TolA II were treated as dummy spheres while TolA III was considered as a rigid body. A generic algorithm was used which then chose an opitimised ensemble containing a group of different TolA II-III conformations whose average theoretical scattering curves fit experimental data well. According to Figure 3.18a, the scattering curve obtained from the optimised ensemble almost perfectly fits the experimental data with a of 1.05. Comparing Rg and Dmax
distribution to the random pool, it displayed Rg and Dmax distribution of optimised
ensemble shifted towards the larger and extended particle with major peaks at Rg = 75 Å and Dmax = 200 Å (Figure 3.18c and d).
Figure 3.18 EOM analysis of TolA II-III. a) SAXS data (symbols) and fitting (line) generated from EOM. b) Distribution of sedimentation coefficient obtained from EOM selected structure (red) comparing with AUC data (blue). Size and shape distributions of the pool structure (black) and selected structure (red) for EOM analysis c) Rg d) Dmax
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The sedimentation coefficient from models generated by EOM was calculated by HYDROPRO and compared to the sedimentation coefficient obtained from the AUC experiment (Figure 3.18b). The s value from models ranged from 1.4 S to 1.8 S but AUC data showed only one species of TolA II-III with an s of 1.5 S. However, EOM analysis is in a good agreement with CRYSOL analysis. EOM predicted that the major species of TolA II-III is at Dmax = 200 Å and model 2
with a maximum length of 190 Å best fitted with experimental data by CRYSOL.