Trayectorias: Primera aproximaci´ on

The H-NS,.g9 construct was produced to investigate the properties o f the region that

appeared to behave independently of the C terminus, and resulted in the line-width broadening effect seen in the NMR spectral data of the full-length protein. This suggested that the H-NS,_g^ polypeptide might have different oligomerisation properties compared to the H-NS,_^ polypeptide, for which NMR spectra did not exhibit the line-width broadening phenomenon (section 3.2.2). The Superose 12 gel filtration traces obtained for H-NS,_g^ at a range of concentrations are shown in Figure 17. The peaks obtained are broad and asymmetric at all protein concentrations and individual, distinct oligomeric states cannot be resolved. This gives the impression that H-NS,_gg adopts a wide range of different self-associated states with a higher average molecular weight at increased protein concentrations. No single, defined state has been reached within the concentration range 8.9 |iM -0.57 mM. A comparison with molecular weight standards results in apparent molecular weights between 24 kD a at 8.9 |liM to 198 kDa at 0.57 mM. These numbers correspond to a change in the most abundant oligomeric state from somewhat larger than a dimer to something in the order o f 2 0-mers within this concentration range.

OD (220 nm) 0.4 0.3 0.2 0.1 30 35 40 45 50 55 60

F ig u re 17. G el f iltra tio n (S u p e ro se 12) stu d ie s o n H-N S,^,. H-NS, ,, at 8.9 ( ), 17.9 ( ), 35.7 ( ), 71.5 ( ), 143 ( ), 286 ( ), and 572 ( ) pM . All experim ents were perform ed in 10 mM K P and 300 mM NaCl at pH 7.0 and 25 °C.

AUC sedimentation equilibrium data obtained for H-NS, gg are shown in Figure 18. These data cannot be accurately modelled for a single species as was possible in the case of H-NS,^ (section 3.2.3). The buoyant molecular mass of this polypeptide was found to vary significantly with the centrifugation speed, ranging from approximately 69 kDa at 8()()() rpm to 31 kDa at 12 000 rpm for a protein

concentration of 0.46 mM (Table 14). The value of M (1-Fp) is expected to remain constant at all speeds for an equilibrium distribution of oligomeric states. These results suggest that H-NS,_gg is able to form homomolecular complexes of considerable size such that when the speed is increased, these will travel to the bottom of the cell and no longer contribute to the analysis. As the speed is reduced, the extent to which the larger components can contribute to the average molecular

weight of the sample increases. Therefore, the overall value of M {\-vp) will also increase, as seen in the case of H-NS, It is important to note that the sample did not exhibit time-dependent aggregation, since the observed masses did not depend on the order of data collection at the different speeds. The ability of H-NS,_g^ to form complexes of considerable size is consistent with the presented gel filtration data. These data clearly describe an oligomerisation behaviour that is dramatically different to that observed for the trimeric H-NS,.^.

(A)

I oxraj

(B) (C)

Radius Radius Radius

Figure 18. A U C sedim entation equilibrium data on H -N S ,„ (0.46 m M ) at different

centrifugation speeds: 8000 rpm (A), 10 0(H) rpm (B), and 12 000 rpm (C ). The experim ents were carried out in 20 m M KP, and 300 m M NaCl a t pH 7.0 and 4 °C. The solid lines represent best fits to equation [3] (see chapter 2) with M (l-ÿ p ) values o f 69367 (A), 46288 (B), and 31448 (C). The residuals o f the fits are shown in each case, dem onstrating that these are random ly distributed around zero.

Table 14. Values for M (l-vp) for H-NS, C ell Speed (rpm ) M i \ - v p ) A 1 8000 7 0 351 2 8000 69 367 3 8000 65 345 B 1 8000 4 6 607 2 8000 4 6 288 3 8000 45 946 C 1 1 2 0 0 0 32 176 2 1 2 0 0 0 31 448 3 1 2 0 0 0 31 287

3.2.5

H-NSg

.j

g is a monomer

The Superose 12 gel filtration traces obtained for are depicted in Figure 19. It is clear that these traces are very similar to those shown for ovalbumin (inset to Figure 13). The peaks are almost completely symmetrical at all protein concentrations, and there is no shifting towards higher or lower molecular weights at any concentration. This suggests that is present in a fixed, single, defined state that is concentration independent. Approximate molecular weights of 9.8 kDa and 10.8 kDa were calculated for at concentrations of 13.4 and 578 uM, respectively, based on a comparison with molecular weight standards (Figure 14). These approximate molecular weights correspond more closely, to a dimer than the expected monomer. However, based on the NM R data obtained for the full-length protein [Figure 10(A)], it is unlikely that any self-association of the C-terminal domain occurs since this would give rise to a similar line-broadening effect as observed for the N-terminus. This discrepancy could be explained if H-NSgg ,^g adopts

C-terminal fragment of H-NS from E. coli by NMR (Shindo et al., 1995). The partially unfolded nature of the H-NSg^.,3^ polypeptide could act to retard the

movement of the protein through the gel matrix, resulting in the estimation of an apparent molecular weight that was higher than expected.

The slightly reduced purity of H-NSgg.,3g (section 2.4.2) could explain the presence of

a few small additional peaks in the gel filtration traces. However, the total lack of concentration dependence of these peaks as well as the major gel filtration peak demonstrate that none of the additional peaks represent a higher-order structure involving H-NSg^ ,3^, and that the impurities do not interact with this polypeptide.

t)D (280 nm) 0.3 0.25 0.2 0.15 0.05 30 35 40 45 50 60 minutes

F ig u re 19. G el filtra tio n (S u p e ro se 12) stu d ie s o n H-NSg, ,3^ a t 13.4 ( ), 26.6 ( ),

52.6 ( ), 105 ( ), 210 ( ), 289 ( ), and 578 ( ) pM in 1.8 mM KP., 10 mM NaP„ 140 mM NaCl and 2.7 m M KCi a t pH 7.4 and 25 °C. The optical density was recorded at 280 nm.

AUC sedimentation equilibrium data on the isolated C-terminal domain,

are shown in Figure 20. These data give an apparent stoichiometry of 1.3, i.e. a value in between a monomer and a dimer. The intermediate stoichiometries may be attributed to possible partial unfolding o f this polypeptide, in agreement with the solution structural data o f a homologous protein in which partial disorder was observed (Shindo et a l, 1995). ce 0.01 S ’. T3 0 .0 0 % -0 .0 1 CE -0 .0 2 (D ü c ce Si o C/> 1 . 1 1 . 0 0 .9 0 . 8 0 .7 0.6 0 .5 0 .4 0 .3 6 .9 7.0 7.1 R a d i u s

F igu re 20. A U C sed im en tation eq u ilib riu m d ata on (96 pM ) at a cen trifu gation speed o f 30 00 0 rpm . T he experim ents w ere carried o ut in 20 m M KPi and 300 m M N aC l at pH 7.0 and 4 °C. T he solid line represents the best-fit to equation [3] (chapter 2) w ith an M { \ - v p ) o f 1703 for this protein solution. T he residuals o f the fit are random ly distributed around zero.

The CD spectra obtained for the C-terminal domain (H-NSgg_,gJ in the peptide backbone region are shown in the inset to Figure 16. It is immediately obvious that these spectra are significantly different to those previously described for the N- terminal domain, H-NS,_g^. In comparison to H-NS,_^, the spectra are relatively weak

previously reported NMR solution structure for the C-terminal domain of H-NS from

E. coli (Shindo et aL, 1995).

There is a negligible concentration dependence shown in the CD spectra obtained for H-NSg9 ,3g at concentrations between 0.32 and 3.2 )iM, which is close to the

experimental uncertainty (inset to Figure 16). This indicates that the C-terminal domain has no involvement in the self-associating mechanism discussed in regards to the N-terminal domain o f the H-NS^^^g protein.