Motores brownianos

H -N S .^

A C-terminally truncated H-NS polypeptide (H-NS, ^^) was constructed to address the issue o f oligomerisation based on the above observations from NMR. This construct provided a soluble polypeptide on which biophysical studies could be performed. A range of ID and 2D NMR spectra of H-NS,.^ demonstrate that this polypeptide can be observed by NMR spectroscopy. A preliminary structural characterisation of this construct reveals that none of the signals observed correspond to those of the full-length H-NS^zos spectra.

The 2D ‘H NOESY spectrum of H-NS, ^^ is shown in Figure 11. The amide region of this spectrum demonstrates the presence of NOE crosspeaks, which are consistent with H-NS,_^ possessing a significant amount of a-helical structure. This is an important observation when taken in conjunction with computer-based secondary structure predictions and the CD spectroscopic study of H-NS,_^ (see below) that demonstrate the predominantly helical nature of this construct and the intimate link which exists between the formation o f a-helix and the self-association of H-NS,.^.

6.5 7.0 7.5 £ Cl X 8.0 8.5 9.0

n i

Figure 11. Am ide region o f a 'H NOESY sp ectrum of H-NS,.#

The results of computer-based structure prediction programs (Lupas et a i , 1991; 1996) suggest it is probable that the first 65 residues of the H-NS^^os protein will adopt a coiled-coil motif (Figure 12). Coiled-coil structures are formed by interactions between a-helical interfaces. It has previously been pointed out that the presence of a characteristic heptad repeat in the N-terminal domain of H-NS, and a sequence similarity to well-known eukaryotic structural coiled-coil proteins such as myosin, a-keratins and dystrophin suggests that the self-association of H-NS could be mediated by interactions between a-helices (Ussery et al., 1994). Furthermore, an

analysis of the sequences of mutant H-NS proteins defective in oligomerisation has been recently reported (Dorman et aL, submitted 1998). These computer predictions reveal that the ability of these H-NS mutants to form a coiled-coil structure will be severely impaired. There appears to be a good correlation between the oligomerisation ability and the predicted potential of these N-terminal residues to form a coiled-coil (Dorman et aL, submitted 1998). The H-NS,^ polypeptide was therefore investigated since this coiled-coil conformation could form the structural basis for self-association.

P rob ab ility 0.8 0.6 0 .4 0.2 20 4 0 6 0 80 100 120 R esid u e n u m b er

Figure 12. Prediction o f coiled-coil structure in the N -term inal parts o f H-NS^.,,,^ using the COILS program (version 2.1; Lupas et a i , 1996). The probability for coiled-coil formation is

reported as a function o f residue num ber in using the scoring matrices M TK ( ) and

M TIDK (~ ). A window of 28 residues was used as recom m ended for predictions o f new coiled-coils. To reduce the possibility o f generating false positives, the program 's w eighting option was em ployed {ie. the two hydrophobic positions a and d o f the heptad repeat are assigned the same weight as the five hydrophilic positions b, c, e , f a n d g).

3.2.3

The N-terminal polypeptide, H-NS,.^^ forms a trimer

Gel filtration was one of the techniques chosen to establish the nature of the oligomeric state adopted by H-NS,^, since it allows an estimation of the approximate molecular weight of the polypeptide. The Superose 12 gel filtration traces obtained for H-NS,^ at concentrations between 9.6 pM and 0.62 mM, in 20 mM KP. and 300 mM NaCl at pH 7 and 25 °C, are depicted in Figure 13. The peaks obtained within this concentration range are all nearly symmetrical and the polypeptide travels through the column at a similar rate, irrespective of concentration. This type of elution profile is expected for a protein which is present in a single, defined state as shown in the inset to Figure 13, which depicts the traces obtained for a monomeric control protein (ovalbumin).

C)D (2 2 0 nm) 1.5 0.6 0 .4 0.2 1.25 1 0 .75 0.5 0 .25 0 45 50 55 60 30 35 40 minutes

Figure 13. Gel filtration (Superose 12) studies on H -N S ,^ . The figure gives the optical density at 220 nm as a function o f retention tim e (minutes). M ain picture; H-NS^^ at 9.6 ( ), 19.3 ( ), 38.5

( ), 77.1 ( ), 155 ( ), 308 (— ), and 617 (— ) pM . The inset gives the traces obtained for a

m onom eric control protein (ovalbum in) at concentrations o f 3 ( ), 6 ( ), 12 (— ), 24 (~ ), and 49

An estimation of the molecular weight of H-NS,^ was obtained by a comparison with the retention times of a mixture of standard proteins of known molecular weights (Figure 14). O D (2 2 0 nm ) 0.1 4.5 0 .0 8 3.5 0 .0 6 40 m inutes 0 .0 4 0.02 0 30 4 0 50 60 7 0 80 m inutes

F ig u re 14. G el filtra tio n (S u p e ro se 12) stu d ies on s ta n d a r d p ro te in s o f k n o w n m o le c u la r

w eight. Main picture; from right to left; vitamin (1.35 kDa), myoglobin (17 kDa), ovalbumin (44

kDa), and immunoglobulin G (158 kDa). There is also an undefined peak corresponding to the void

volume and thyroglobulin (670 kDa), but this is not used in the analysis. The inset gives the linear

regression fit obtained by plotting the log,,, molecular weight (Mw) of the standards as a function of

time (minutes). This gave an R value of 0.998.

The approximate molecular weights determined from the standard curve (inset to Figure 14) for H -N S,^ at concentrations of 9.6 and 617 pM, were 26 kDa and 28 kDa, respectively. The molecular weight of an H-NS, monomer is 7959 Da (see section 2.4.3), therefore, at all concentrations shown, these values correspond to an oligomeric state which is in between that of a trimer and a tetramer. These results are also consistent with H-NS,,^ adopting a defined oligomeric state under these

conditions. It was, however, noted that at the lowest protein concentrations, the peaks exhibited a slight tendency to shift towards lower molecular weights (Figure 13). This behaviour is consistent with the suggestion that the self-association reaches a limit {i.e. the size of the oligomer no longer increases) at higher concentrations (>32 |xM), and that dissociation will occur upon further dilution.

Analytical ultracentrifugation (AUC) sedimentation equilibrium experiments were performed in order to obtain an independent measure o f the molecular weight of the H-NS,.^ complexes in solution. A representative data set is presented in Figure 15. The experiments were run at three different speeds using protein concentrations of 0.43 and 0.57 mM (similar to NMR concentrations). An average molecular weight of 22.6 ± 0.6 kDa was calculated based on these data using v = 0.7294 cm^ g ‘ and p =

1.0134 g cm^ (see Chapter 2). As the H-NS,.^ monomer has a molecular weight of 7959 Da (see section 2.4.3), the above molecular weight corresponds closely to that of an H-NS,.g4 trimer, which is in good agreement with the approximate value

obtained by gel filtration (between a trimer and a tetramer) at significantly lower protein concentrations.

CD spectropolarimetry provides a means to examine the structural basis for self­ association of the N-terminal domain of H-NS(,2os- The CD spectra obtained for H-

NS,_^ at concentrations ranging from 0.33 to 166 pM are shown in Figure 16. The magnitude of the CD in the peptide backbone region (185-260 nm) is strongly concentration dependent within this range in 10 mM KP^ and 10 mM NaCl at pH 7.0 and 25 °C, however, at concentrations above 33 |xM no further changes in the CD spectra were observed.

The shape of the CD spectra, in particular the two negative maxima close to 208 and 220 nm and the positive maximum at close to 192 nm, signify the presence of a considerable amount of a-helical structure, that increases with higher protein concentrations. The result of a deconvolution of the spectra in terms of a-helical type structure content is given in Table 13.

w 0.01 -0.01 Q) - 0 .0 2 0 .9 S 0 .7 o

^

R adiu s

F ig u re 15. A U C se d im e n ta tio n e q u ilib riu m d a ta on H -N S ,^ (0.57 m M ) a t a c e n trifu g a tio n sp e ed o f 15 500 rp m . The experiment was carried out in 20 mM KR and 300

mM NaCl at pH 7.0 and 4 °C. The solid line represents the best-fit to equation [3] (see Chapter

2) with an M (l-i7p) value of 5876 for this protein solution. The residuals of the fit are

A e ( M ^cm 10 7 .5 5 -2.5 2 .5 0 - 2 . 5 - 5 1 90 200 210 220 2 3 0 2 4 0 2 5 0 2 6 0 A (nm)

F ig u re 16. C D stu d ie s on a n d Tlie figure shows the delta epsilon (Ae) as a

function o f waveiengtii (A) in the peptide backbone region (1 8 5 -2 6 0 nm). M ain picture; H -N S ,^ at 0.33 ( ), 3.3 ( ), 33 ( ), and 166 { ) pM in 10 mM KP, mid 10 mM NaCl at pH 7.0 and 25 °C. The inset gives the spectrum o f H -N S,, ,3^ at 0.32 ( ), 3.2 ( ), 32 ( ), and 75.5 ( ~ ) pM in 5 mM

KP, and 10 mM NaCl at pH 7.0 and 25 °C. The C -term inal part o f the protein gives a com paratively w eak C D spectrum. Tlie sm all changes in tlie CD spectrum o f H -N S,, ,3,5 witli concentration are

Table 13. Deconvolution of the circular dichroism data.

Protein C o ncentration a -h e lix

(p M ) (% ) H-NS„os 310 45.3“ 31 47 ” (44.3') 3.1 30.4” (34.3“) 0.31 14.8” 31 19 (91 °C) 3.1 _{11 (95 °C)} 3.1 _{48.1“ (25 °C)} 3.1 63.9“ ( - 6 6 °C) H - N S ,, ” 166 55 33 53.2 3.3 34.9 0.33 19.4 33 19.3 (74 °C) 3.3 _{13.2 (73 °C)} H -N Sj,.,3/ 75.5 10.7 32 10.5 3.2 9.6 0.32 7.8

in 10 m M KP^ and 300 m M N aC l at pH 7.0 and room tem perature, in 10 m M K Pjand 10 m M N aC l at pH 7.0 and room tem perature, in 5 m M KP; and 10 m M N aC l at pH 7.0 and room tem perature, in 5 m M KP., 10 m M N aC l and 70 % ethanediol at pH 7.0.

These results demonstrate that the N-terminal 64 amino acids of H-NS^jos capable of oligomerisation. self-associates upon increased concentration and the self-association is coupled to the formation of an increasing amount o f a-helical secondary structure until a limiting concentration is reached at which secondary structural changes cease to be observed (Figure 16). The change in a-helical content when the concentration of H-NSj ^^ is raised from 0.33-33 \iM is approximately 34 %; however, subsequently increasing the concentration to 166 |xm only gives rise to a 2 % change. The observed increase in a-helical structure as a function of concentration is consistent with the coiled-coil structure predictions; hence, a coiled-

coil structure could form the basis for the oligomerisation of the H-NS,_^ polypeptide.

In contrast to its behaviour in the context of the intact protein, gives measurable NM R spectra (Figure 11). The limited line broadening and relaxation data are consistent with H-NS, ^^ adopting a defined state which is much smaller than the oligomeric state adopted by the full-length H-NS^^^osprotein (section 3.2.6).