Dynamics of the Escherichia coli O91 O-Antigen Polysaccharide in

Mar 11, 2004 - A model-free analysis of the relaxation data revealed a complex dynamical behavior where the sugar residues could be described by a glo...
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Biomacromolecules 2004, 5, 1015-1020

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Dynamics of the Escherichia coli O91 O-Antigen Polysaccharide in Solution as Studied by Carbon-13 NMR Relaxation Kristina Lycknert and Go¨ran Widmalm* Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden Received December 8, 2003; Revised Manuscript Received February 9, 2004

The dynamics of the O-antigen part of the lipopolysaccharide from the enterohemorrhagic Escherichia coli O91 has been determined in solution using 13C NMR relaxation measurements at two magnetic field strengths, 9.4 and 14.1 T, thereby facilitating the testing of several dynamical models. The biological repeating unit, consisting of five sugar residues and substituents, could be determined by spectral analysis of different 1 13 H, C correlations and corroborated by the relaxation data. The site specifically 13C-labeled material was shown to have ∼10 repeating units with a narrow distribution. A model-free analysis of the relaxation data revealed a complex dynamical behavior where the sugar residues could be described by a global correlation time (τm ) 5.4 ns), generalized order parameters (S2 ≈ 0.63), and different correlation times for internal motions related to their position in the repeating unit along the polymer (τe ≈ 360-520 ps). One of the sugar residues showed, in addition, a chemical exchange contribution. Furthermore, a substituent on another sugar residue was described by two order parameters (Sf2 ) 0.51 and Ss2 ) 0.21). The solution dynamics of the polysaccharide are thus described by highly intricate motions, both in amplitude and time scales. These results are of significance in the general description of polysaccharides surrounding bacterial cell surfaces and in the presentation of antigenic epitopes to the immune system of an invaded host. Introduction Polysaccharides play many important roles in nature ranging from structural support as cellulose, energy storage in the form of amylose, and coating of bacterial cell surfaces to being parts of tissues and fluids in animals and man. The relationship between structure and pysico-chemical properties can be studied with a variety of techniques such as dynamic light scattering,1 viscometry,2 or nuclear magnetic resonance spectroscopy.3-8 The latter technique has been used extensively, also together with molecular modeling, to investigate dynamics of polysaccharides and oligosaccharide fragments thereof. In some of these studies, molecular modeling has played an essential part in the interpretation of experimental data, whereas in other cases, molecular modeling has been the main tool, complemented by other techniques.9-12 Gram-negative bacteria have lipopolysaccharides (LPS) anchored in their outer membrane. Besides the lipid A and core parts, the LPS consists of an O-antigenic polysaccharide, specific for each strain, which usually constitutes 2-7 sugar residues in the repeating unit. These O-antigens determine which serogroup that a bacterium is classified into. In addition, the H-antigen (flagella) determines the serotype and capsules may also be present, hence these are described as K-antigens. To date, ∼180 different serogroups of the E. coli species have been typed with immunological methods, with strain numbers being given consecutively as novel strains are being characterized. More recently, an alternative * To whom correspondence should be addressed. E-mail: [email protected].

typing scheme has been proposed based on virulence factors, known as virotyping.13 One of these virotypes is enterohemorrhagic E. coli (EHEC) that produces a Shiga-like toxin and an enterohemolysin. The EHEC strains cause hemorrhagic colitis that can lead to severe complications in man including acute kidney failure. Members of this group are the well-known E. coli O157:H7 as well as E. coli O26 and E. coli O91. The structure of the O-antigen polysaccharide from E. coli O91 was recently determined14 and was shown to have pentasaccharide repeating units with the structure: f4)-RD-Quip-3-N-[(R)-3-hydroxybutyramido]-(1f4)-β-D-Galp(1f4)-β-D-GlcpNAc-(1f4)-β-D-GlcpA-6-N-Gly-(1f3)-βD-GlcpNAc-(1f. In addition to the standard growth conditions for the bacterium to give material for chemical analysis, the strain was grown with the addition of D-[UL-13C]glucose which facilitated assignments of NMR resonances and sequence determination. Additional cultures with specifically labeled D-[1-13C]glucose or D-[6-13C]glucose shed light on biosynthetic pathways present in the bacterium. To examine the flexibility and dynamics of the E. coli O91 O-antigen polysaccharide, we herein utilize the material grown on D-[1-13C]glucose which due to direct incorporation of the specifically labeled glucose results in 13C-enrichment at the anomeric positions of the sugar residues in the repeating unit and as a result of catabolism leads to specific labeling in substituent groups such as the (R)-3-hydroxybutyramido group. 13C relaxation data were obtained at two magnetic field strengths and the results are interpreted with

10.1021/bm0345108 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/11/2004

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different models within the framework of the model-free analysis.15 Materials and Methods Sample Preparation. The lipid-free polysaccharide grown on specifically labeled D-[1-13C]Glc was used for the NMR relaxation studies. The polysaccharide was dissolved in D2O to give a ∼7 mM solution with respect to a specifically labeled 13C repeating unit in the polymer. The sample was treated with CHELEX-100, filtered, and transferred to a 5 mm NMR tube. It was degassed by several freeze-pumpthaw cycles before being flame-sealed under vacuum. Mass Spectrometry. The MALDI time-of-flight spectrum was acquired in the positive mode on an Autoflex (Bruker Daltonik GmbH, Bremen, Germany) with dihydroxybenzoic acid as a matrix. NMR Spectroscopy. Carbon-13 relaxation measurements were performed at 310 K using Varian Inova 400 (9.4 T, 1H frequency 399.511 MHz) and Varian Inova 600 (14.1 T, 1H frequency 599.748 MHz) NMR spectrometers. The carrier frequency of the 13C observe nucleus was placed at ∼65 ppm and the spectral window covered 120 ppm. Off-resonance effects16 were investigated by placing the carrier frequency in the regions where signals were resonating, but no significant change was observed, and the carrier was subsequently not altered. In all experiments, WALTZ broadband proton decoupling was carried out during the acquisition period. Standard temperature equipment provided by the manufacturer was used. Special care was taken to reduce any systematic difference due to the temperature on the two spectrometers, which was checked by an electronic thermometer inserted into the magnets and by an ethylene glycol thermometric solution.17 T1 was measured with the inversion-recovery method using 10 τ values between the inversion pulse and the read pulse. The number of scans was 2960 and 1408 for each τ value at the lower and higher magnetic field, respectively. T2 was measured with the Carr-Purcell-Meiboom-Gill sequence using 10 τ values as described,18 with the delay between π pulses set to 500 µs. The number of scans was 2048 for each τ value at the two magnetic fields. Heteronuclear NOE was measured by the dynamic NOE technique with reduced decoupling power during the NOE buildup time using one long period (>5 T1) and one short period (1 ms). The NOE factors (1 + η) were evaluated by taking the ratio between peak intensities obtained with the long and the short period. The recovery delay was >10 T1. The number of scans was 4096 for each irradiation period at the two magnetic fields. Prior the Fourier transformation, the FID was multiplied with an exponential line broadening factor of 3 Hz. The T1 and T2 values were evaluated using nonlinear fitting supplied in the VNMR software (Varian). All experiments were carried out 5 times from which the mean values, their standard deviations, and subsequently the 95% confidence intervals were calculated. Relaxation Data Analysis. The relaxation of protonbearing carbon-13 nuclei is dominated by the dipole-dipole interactions with neighboring protons. For carbohydrate

Lycknert and Widmalm

systems, the chemical shift anisotropy (CSA) mechanism is relatively small but not necessary negligible. The relaxation parameters can be expressed in terms of spectral density functions taken at different combinations of the carbon (ωC) and proton (ωH) Larmor frequencies. The rates for relaxation of longitudinal and transverse magnetization (R1 and R2, respectively) and the 1H,13C cross-relaxation effect (NOE) are given by19,20 R1 ) T1-1 )

d2 [J(ωH - ωC) + 3J(ωC) + 4 6J(ωH + ωC)] + c2J(ωC) (1)

R2 ) T2-1 )

d2 [4J(0) + J(ωH - ωC) + 3J(ωC) + 6J(ωH) + 8 c2 6J(ωH + ωC)] + [4J(0) + 3J(ωC)] + Rex (2) 6

NOE ) 1 +

d 2 γH [6J(ωH + ωC) - J(ωH - ωC)] (3) 4R1 γC

in which T1 and T2 are the relaxation times of longitudinal and transverse magnetization, respectively, d ) ({µ0}/ {4π})γCγHprCH-3, c ) {ωC∆σ}/{x3}, µ0 is the permittivity of free space, γC and γH are the magnetogyric ratios for carbon and proton, respectively, p is Planck’s constant divided by 2π, rCH ) 1.117 Å is the carbon-proton bond length,21 and ∆σ ) 30 ppm is the CSA value appropriate for carbohydrate systems such as these investigated herein.22 For carbons with two protons directly attached, such as C2 of the substituent at residue E, the analysis is performed on a per CH pair basis; that is, R1 and R2 are divided by a factor of 2, whereas the NOE remains the same. The Rex term in eq 2 is often included to account for chemical or conformational exchange.20 Interpretation of the relaxation data as amplitudes and time scales often employ the model-free formalism pioneered by Lipari and Szabo,15 and extend by Clore and co-workers,23 in which the spectral density function, J(ω), is modeled as

[

]

(Sf2 - S2)τ S2τm 2 + J(ω) ) 5 1 + (ωτ )2 1 + (ωτ)2 m

(4)

where τ-1 ) τm-1 + τe-1, τm is the correlation time for the global motion, common to the whole molecule, τe is the correlation time for internal motions, S2 is the square of the generalized order parameter which reflects the spatial restriction of local motion, and Sf2 and Ss2 are the squares of the order parameters for the internal motions on the fast and the slow time scales, respectively, with S2 ) Sf2 Ss2. The correlation times for fast and slow internal motions are described by τf and τs, respectively. Fitting of the relaxation rates to model-free parameters was performed with the program Modelfree,20 version 4.15, running on a PC under Linux. In the Modelfree program, different models can be assessed and besides the fitting of a global correlation time, τm, the following parameters are fitted: (1) S2, (2) S2 and τe, (3) S2 and Rex, (4) S2, τe, and Rex, and (5) Sf2, Ss2, and τe. In models 1-4, Sf2 ) 1 and S2 ) Ss2. In models 2 and 4, τe )

Dynamics of E. coli O91 O-Polysaccharide

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Figure 1. Schematic of the O-antigen polysaccharide from Escherichia coli O91 having n repeating units. Sugars residues are denoted by A-E and the substituent on the latter residue by SE.

Figure 2. Anomeric region of the 13C NMR spectrum with annotated resonances corresponding to the respective sugar residues. The low intensity E′ signal corresponds to the terminal nonreducing residue in the polysaccharide.

τf, whereas in model 5, τe ) τs. The residual sum-squared error and the F statistic, both at a confidence level R ) 0.05, were used to assess whether a model adequately described the data and an improvement in the fit to a more complicated model is significant, respectively. In the analysis of the relaxation data, it was evident that the statistical errors of the standard deviations calculated from experiment were too limited and an estimate of the experimental uncertainties associated with the different residues led to an increase by a factor of 2.5, except for residue D for which and additional unit was added leading to a factor of 3.5. Results The structure of the repeating units in the O-polysaccharide from E. coli O91 is shown in Figure 1. It consists of 5 sugar residues, all being linked through a secondary carbon of the pyranoid sugar rings. The material used in this study was grown on specifically labeled D-[1-13C]Glc which lead to site specific 13C labeling at the anomeric positions of residues A through E as well as in the substituent SE which is an (R)-3-hydroxybutyramido group. The anomeric region of the 13 C NMR spectrum shows the corresponding resonances (Figure 2). In addition, a signal of low intensity was observed at δC 100.2, being ∼10% of that from residue E at δC 99.8. A coupled 1H,13C-HSQC spectrum showed that the small signal has the same spectral characteristics as that of the anomeric resonance of residue E, viz., δH 4.90, 1JC,H ) 173 Hz, and 3JH,H ) 3.5 Hz. Thus, the minor signal should originate from a residue that has a slightly different environment compared to E in the polymer. Interestingly, an almost equal relative ratio was observed for the signal from the SE residue and an adjacent one at δC 46.1. Again the observation occurs at residue E. Furthermore, a 1H,13C-HMBC spectrum

revealed a correlation between the anomeric carbon resonance at δC 100.2 and a proton resonance at δH 4.05, closely similar to that between C1 in residue E and H4 in residue A. These results strongly suggest that the signal from the minor component arises from residue E as the terminal component, denoted E′, in the polymer chain and not as the residue being linked to the outer core region. Thus, the biological repeating unit should consequently be defined as E-A-D-B-C-, as shown in Figure 1. From integration of the minor 13C signals to those of the anomeric resonance of residue E and the methylene carbon of SE, it is evident that the polysaccharide consists of ∼10 repeating units in the O-antigen part. Analysis of the E. coli O91 PS by MALDI-TOF MS revealed a narrow distribution of the major peaks centered around 11.1 kDa, which corresponds to 9 ( 1 repeating units (when the core sugars that remain after acidic delipidation of the LPS have been accounted for). Thus, the results from two independent techniques are highly consistent in describing the degree of polymerization of the E. coli O91 PS. Knowing the biological repeating unit, the number of repeating units in the O-polysaccharide, and the distribution of these repeating units, we now turn to the study of the polysaccharide dynamics. Relaxation measurements were performed on the resonances from anomeric carbons as well as a methylene carbon (C2 of SE). The anomeric region in the 13C NMR spectrum has resolved signals. However, the spectral overlap in the 1H,13C-HSQC spectrum is severe. Consequently, the 13C relaxation measurements were performed as 1D 13C direct detection, which we often have used for oligosaccharides.18,24 Longitudinal T1 and T2 transverse relaxation times complemented with 1H,13C NOE were measured at two magnetic field strengths and the results are presented in Table 1. It is evident that the relaxation times are different in several of the residues of the polysaccharide and that the substituent behaves in another way compared to the sugar residues in the polymer backbone. The magnetic field dependence is also readily observed. The relaxation data for residue E′ were also analyzed to investigate any trends present. Compared to E, the heteronuclear NOE of E′ was larger and T2 was longer supporting the notion that more rapid dynamics are present at the terminus of the chain. Interestingly, at the higher magnetic field, T1 of E′ was shorter than for E, whereas at the lower magnetic field, a longer relaxation time was observed. This reveals that the relaxation times are close to the T1 minima at the two magnetic field strengths. Taken together, the relaxation data

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Table 1.

13C

Lycknert and Widmalm

Relaxation Data for the E. coli O91 O-Polysaccharide

B0/T

residue A B C D E SE A B C D E SE

9.4

14.1

R1/s-1

T1/ms (3.0)a

[0.06]b

315 327 (4.5) 302 (1.8) 332 (4.5) 288 (5.6) 286 (4.8) 484 (8.7) 503 (9.8) 467 (7.2) 507 (6.1) 461 (7.2) 382 (6.0)

3.17 3.06 [0.08] 3.32 [0.03] 3.01 [0.12] 3.48 [0.14] 1.75 [0.06] 2.07 [0.08] 1.99 [0.08] 2.14 [0.07] 1.97 [0.07] 2.17 [0.07] 1.31 [0.04]

T2/ms

R2/s-1

NOE

56.7 (4.5) 66.6 (2.5) 50.2 (3.1) 69.4 (6.3) 58.6 (5.3) 119.1 (12.4) 68.2 (1.0) 71.6 (1.9) 48.4 (1.0) 78.4 (2.8) 62.1 (1.6) 150.8 (8.5)

17.69 [2.70] 15.04 [1.12] 19.97 [2.50] 14.47 [3.69] 17.15 [3.21] 4.22 [0.86] 14.66 [0.44] 13.96 [0.75] 20.66 [0.87] 12.77 [1.25] 16.09 [0.87] 3.32 [0.38]

1.67 (0.013) [0.040] 1.64 (0.015) [0.048] 1.60 (0.025) [0.078] 1.65 (0.008) [0.038] 1.53 (0.011) [0.035] 2.16 (0.028) [0.070] 1.58 (0.009) [0.028] 1.58 (0.008) [0.025] 1.52 (0.004) [0.012] 1.60 (0.007) [0.028] 1.48 (0.004) [0.012] 2.06 (0.015) [0.038]

a The 95% confidence interval is shown in parentheses. b Estimated experimental uncertainties in the relaxation parameters are given in square brackets.

Figure 3. Relaxation parameters for the different residues obtained at 14.1 T: (a) Longitudinal relaxation rate, (b) transverse relaxation rate, and (c) heteronuclear NOE. The error-bars show the experimental uncertainties that were used in the model-free fitting. Table 2. Dynamical Parameters for the E. coli O91 O-Polysaccharide with τm ) 5.42 ns residue

model

S2

A B C D E SE

2 2 4 2 2 5

0.63 (0.02)a 0.64 (0.02) 0.62 (0.01) 0.64 (0.02) 0.62 (0.02) 0.11b (0.02)

Sf2

τe/ps

0.51 (0.02)

387c (20) 376 (21) 455c (11) 366 (23) 517 (20) 283d (15)

Rex/s-1

2.7e (0.4)

a The error as one standard deviation in the fitted parameter is shown in parentheses. b S2 ) Sf2 Ss2. c τe ) τf. d τe ) τs. e Exchange term at 9.4 T.

corroborate the conclusion that E′ is the terminal residue in the polymer, and this residue can therefore readily be omitted from the analysis of the internal dynamics of the polysaccharide. The relaxation rates, and associated experimental uncertainties, were calculated (Figure 3 and Table 2) and used to obtain motional parameters as described below. For a model-free interpretation of the relaxation data fitting was performed to the different models described above. An initial trial revealed that a description of the dynamics by τm and S2 only was insufficient for the backbone sugar residues. However, using in addition a correlation time for internal motions, τe, i.e., model 2, gave good results for all sugar residues but one, viz., residue C. For comparison, each residue was also fitted with a local correlation time, τloc, which gave highly similar results for all residues but C, which showed a τloc twice as long as the other residues. Such an interpretation seems highly unreasonable and unphysical

Figure 4. Effective correlation time for internal motions, τe, of the sugar residues as a function of their position in the repeating unit of the polymer. The filled diamond depicts that this residue also exhibits a slow process due to conformational exchange. The circle displays τs of the substituent SE. The error-bars show one standard deviation in the fitted parameter.

since the residues are present like beads on a string. Model 3 in which an Rex term has been added to the first model behaved essentially as badly as model 1. Most interestingly, when the Rex term was added to model 2, resulting in model 4, the C residue behaved well with a significant exchange term present. When the substituent SE was tested with the above models, τm became significantly shorter, by in some cases almost an order of magnitude. However, SE could indeed be fitted to model 5 that has two order parameters, one for fast and one for slow motions. Thus, we now performed a combined fit of the relaxation rates to models 2, 4, and 5 depending on residue. The results are shown in Table 2. The global correlation time τm ) 5.4 ns and the amplitudes of the molecular motions described by S2 ≈ 0.63 being highly similar for all of the sugars that indeed are linked through secondary carbons of the adjacent residues. The correlation times for internal motions, τe, are found in the range 360520 ps, i.e., an order of magnitude shorter than τm. It is evident that there is a smooth change in the internal dynamics along the polysaccharide chain (Figure 4). Such a behavior is also indicated from R1 and NOE data (Figure 3). The R2 data suggest, though, that an additional contribution to the relaxation may be present, a finding that was confirmed through the fitting to model 4 that contains a chemical exchange term for residue C. The results are interpreted as slow conformational dynamics with Rex ≈ 6 s-1 at the higher magnetic field. The extended model-free approach has been

Dynamics of E. coli O91 O-Polysaccharide

used in the analysis of protein dynamics and could successfully be applied to the dynamics of the methylene group in the (R)-3-hydroxybutyramido substituent. For SE, which has τf f 0 we obtained Sf2 ) 0.51, Ss2 ) 0.21, and τs ) 283 ps. The latter correlation time is of the same magnitude as those of the sugar residues in the backbone chain of the polymer, whereas the amplitude of the dynamics shows an extremely flexible substituent having S2 ) 0.11. This analysis shows that complex motions of different amplitudes and time scales are present in the O-antigen part of this bacterial LPS. Discussion We have previously been able to estimate the number of repeating units in some O-antigen polysaccharides as well as suggest the biological repeating unit in the O-polysaccharide. The O-antigen polysaccharide from E. coli O35 was shown to consist of ∼8 repeating units from integration of amide protons25 and the O-antigen polysaccharide from E. coli O8, which has a terminal Man3Me sugar residue, consists of ∼10 repeating units from analysis of 13C NMR data.26 A longer O-antigen polysaccharide is present in E. coli O159 having ∼23 repeating units as determined by mass spectrometry.27 E. coli O91, with its O-antigen having ∼10 repeating units, is therefore similar in this respect to other E. coli bacteria producing O-antigens with several repeating units. From the results reported in the literature, most O-polysaccharides are being polymerized via transfer of the growing chain to a newly synthesized repeating unit on undecaprenolphosphate, a membrane-bound carrier.28 This pathway is known as the Wzy-dependent one in which the biological repeating units are first synthesized at the cytoplasmic face of the plasma membrane and then transferred en bloc to the periplasmic face of the membrane. Subsequently, growth of the polymer occurs at the reducing end of the polymer. Two other biosynthetic pathways have been described. The ABC transporter-dependent pathway, in which the polysaccharide is formed by transfer of glycosyl residues to the nonreducing end, which takes place at the cytoplasmic face of the plasma membrane, followed by subsequent transfer to the periplasmic face. An example of this pathway is the synthesis of the E. coli O8 O-antigen polysaccharide, a mannose containing homopolymer. The synthase-dependent pathway has only been described for one system, viz., the O:54 O-polysaccharide of Salmonella,29 a homopolymer consisting of N-acetylmannosamine. Again the growth of the polymer chain occurs at the nonreducing terminus, but in this case, the polymer is simultaneously being elongated and extruded across the plasma membrane. In the present investigation, all data support the presence of residue E as the terminal one. Consequently, the biological repeating unit is thus defined. The sugar residue at the reducing end of the repeating unit is an N-acetylglucosamine. This finding is interesting since it was demonstrated that for the E. coli O7 O-antigen polysaccharide the first sugar unit to be added to the lipid acceptor was GlcNAc, thereby defining the biological pentasaccharide repeating unit.30 Furthermore, it was suggested that in the O18, O75 and O111

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O-antigen polysaccharides from E. coli, the single Nacetylglucosamine residue in the repeating unit is the first sugar to be added to the lipid carrier. We anticipate that the biosynthetic pathway that is utilized by E. coli O91 for producing its O-antigen is the Wzy-dependent one. The addition of a GlcNAc residue as the first sugar unit of the biological repeating unit is probably a common motif,28 although not necessarily the sole possibility for these polymers. These ideas are further substantiated by the fact that, in the biosynthesis of the Enterobacterial common antigen, the GlcNAc residue in the trisaccharide repeating unit is added as the first sugar residue to the membrane-bound carrier.31 Given that a narrow distribution for the number of repeating units was observed, the smooth type LPS should be modulated by a chain-length determinant, like Wzz, since in its absence the amount of a certain O-antigen chain length is inversely proportional to the chain length per se. In light of the above results, detailed elucidation of the biosynthetic pathway of the E. coli O91 O-antigen polysaccharide poses an interesting problem that can now be addressed in a fruitful way. The choice of an appropriate model for the description of protein dynamics is still a matter of intense enquiry, and novel ways in which relaxation data can be interpreted are continuously being put forward.32-37 Polysaccharide dynamics are as complex as those of polypeptides and proteins, and some insights come from techniques related to random coils dynamics in general.38 In the field of polysaccharide dynamics, Bush and co-workers have performed heteronuclear relaxation experiments on, inter alia, 13C-enriched samples of a cell wall polysaccharide of Streptococcus mitis J22, at a single magnetic field.39,40 They found for the polymer with heptasaccharide repeating units, inter alia, a global correlation time of a few ns, S2 ≈ 0.6-0.9, and short correlation times for internal motions, τe < 150 ps. In the work of Henderson et al. on R(2f8)-linked polysialic acids, neither a rigid isotropic rotor nor a rigid helix could account for the 13C relaxation data, which were measured at two magnetic fields.41 However, a distribution of correlation times, on the order of a few ns, could satisfactory describe the dynamics which are consistent with internal and segmental motions, describing a random coil in solution. Moreover, when a sample with ∼40 sugar residues, which is similar in chain length to the E. coli O91 polysaccharide, was compared to a polysialic acid polymer in which the number of sugar residues were an order of magnitude larger, no significant change in the motional parameters could be supported. In addition, the effect on viscosity of the samples on relaxation parameters, investigated with a 13C-enriched sample, was negligible. Both results argue for the presence of internal motions as a predominant source of the observed relaxation data. The results from the E. coli O91 O-antigen corroborate the previous findings that flexibility is an integral part in the description of a polymer. Our present findings reveal in further detail different dynamics along the polymer chain. This may be of fundamental importance in the interaction with other molecules, in particular proteins, since not only is an immunodominant sugar necessary but a recent

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investigation using a monoclonal antibody has shown that the antigenic epitope corresponds to the repeating unit consisting of three sugar residues in the cell-wall polysaccharide of Streptococcus group A.42 The structure of E. coli O91 O-antigen can be viewed as a long stretch of residues over the whole repeating unit with a bend at the EfA glycosidic linkage in which an R-linked residue substitutes the 4-position of a residue with the galacto-configuration. The corresponding stereochemical arrangement is present in the LPS from Salmonella dysenteriae type 2, which in the latter case is also suggested as a mimotype, which allows the bacterium to evade the immune system of the host.43 A significant part of the flexibility at a glycosidic linkage can be attributed to the fact that for the ψ torsion angle an elongated minimum energy well is present43 or that interconversion occurs between two conformational states for this degree of freedom. In addition, it is well-known that anti-conformational states at the glycosidic linkage are present for oligosaccharides44,45 and that to some extent this may also occur in larger oligomers being models for polysaccharides.46 The contribution to R2 in the form of a chemical exchange term could be interpreted as slow conformational interconversion between such conformational states on the µs to ms time scale revealing further complexity in the dynamics of the polysaccharide. In conclusion, we have described the flexibility and dynamics of the E. coli O91 O-antigen in detail. Complex motions are present both in magnitude and time scales ranging from very short on the order of ps, to long possibly up to ms. Not only was the biological repeating unit determined but also the number of repeating units in the polymer could be ascertained with a narrow distribution. Further investigations should be directed along the lines of molecular dynamics simulations and hydrodynamics calculations; methods that have been shown to be powerful in the analysis of oligosaccharide conformation and dynamics.24,47 Acknowledgment. This work was supported by a grant from the Swedish Research Council. We thank Dr. Franz Mayer-Posner, Bruker Daltonik GmbH, Bremen, Germany, for providing the MALDI-TOF MS spectrum and Dr. Lena Ma¨ler for stimulating discussions. References and Notes (1) Liu, J. H.-Y.; Brant, D. A.; Kitamura, S.; Kajiwara, K.; Mimura, M. Macromolecules 1999, 32, 8611. (2) Yang, Z.; Huttunen, E.; Staaf, M.; Widmalm, G.; Tenhu, H. Int. Dairy J. 1999, 9, 631. (3) Brant, D. A.; Liu, H.-S.; Zhu, Z. S. Carbohydr. Res. 1995, 278, 11. (4) Cowman, M. K.; Hittner, D. M.; Feder-Davis, J. Macromolecules 1996, 29, 2894. (5) Brant, D. A. Pure Appl. Chem. 1997, 69, 1885. (6) Cavalieri, F.; Chiessi, E.; Paci, M.; Paradossi, G.; Flaibani, A.; Cesa`ro, A. Macromolecules 2001, 34, 99. (7) Cowman, M. K.; Feder-Davis, J.; Hittner, D. M. Macromolecules 2001, 34, 110. (8) NMR spectroscopy of polymers in solution and in the solid state; Cheng, H. N., English, A. D., Eds.; ACS Symposium series 834; American Chemical Society, Washington, DC, 2003.

Lycknert and Widmalm (9) Gagnaire, D.; Pe´rez, S.; Tran, V. Makromol. Chem. 1984, 185, 829. (10) Kotra, L. P.; Golemi, D.; Amor, N. A.; Liu, G.-Y.; Mobashery, S. J. Am. Chem. Soc. 1999, 121, 8707. (11) Paradossi, G.; Cavalieri, F.; Chiessi, E., Macromolecules 2002, 35, 6404. (12) Faber, E. J.; van Kuik, J. A.; Kamerling, J. P.; Vliegenthart, J. F. G. Biopolymers 2002, 63, 66. (13) Salyers, A. A.; Whitt, D. D. Bacterial Pathogenesis: A Molecular Approach; ASM Press: Washington, DC, 1994; p 190. (14) Kjellberg, A.; Weintraub, A.; Widmalm, G. Biochemistry 1999, 38, 12205. (15) Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4546. (16) Korzhnev, D. M.; Tischenko, E. V.; Arseniev, A. S. J. Biomol. NMR 2000, 17, 231. (17) Ammann, C.; Meier, P.; Merbach, A. E. J. Magn. Reson. 1982, 46, 319. (18) Ma¨ler, L.; Widmalm, G.; Kowalewski, J. J. Biomol. NMR 1996, 7, 1. (19) Doddrell, D.; Glushko, V.; Allerhand, A. J. Chem. Phys. 1972, 56, 3683. (20) Mandel, A. M.; Akke, M.; Palmer, A. G., III. J. Mol. Biol. 1995, 246, 144. (21) Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1998, 120, 12334. (22) Ko¨ve´r, K. E.; Batta, G. J. Magn. Reson. 2001, 150, 137. (23) Clore, G. M.; Szabo, A.; Bax, A.; Kay, L. E.; Driscoll, P. C.; Gronenborn, A. M. J. Am. Chem. Soc. 1990, 112, 4989. (24) Rundlo¨f, T.; Venable, R. M.; Pastor, R. W.; Kowalewski, J.; Widmalm, G. J. Am. Chem. Soc. 1999, 121, 11847. (25) Rundlo¨f, T.; Weintraub, A.; Widmalm, G. Eur. J. Biochem. 1998, 258, 139. (26) Jansson, P.-E.; Lo¨nngren, J.; Widmalm, G.; Leontein, K.; Slettengren, K.; Svenson, S. B.; Wrangsell, G.; Dell, A.; Tiller, P. R. Carbohydr. Res. 1985, 145, 59. (27) Linnerborg, M.; Weintraub, A.; Widmalm, G. Eur. J. Biochem. 1998, 266, 246. (28) Keenleyside, W. J.; Whitfield, C. In Endotoxin in Health and Disease; Brade, H., Opal, S. M., Vogel, S. N., Morrison, D. C., Eds.; Marcel Dekker: New York, 1999; p 331. (29) Keenleyside, W. J.; Whitfield, C. J. Biol. Chem. 1996, 271, 28581. (30) Alexander, D. C.; Valvano, M. A. J. Bacteriol. 1994, 176, 7079. (31) Erbel, P. J. A.; Barr, K.; Gao, N.; Gerwig, G. J.; Rick, P. D.; Gardner, K. H. J. Bacteriol. 2003, 185, 1995. (32) Daragan, V. A.; Mayo, K. H. Prog. NMR Spectrosc. 1997, 31, 63. (33) Philippopoulos, M.; Mandel, A. M.; Palmer, III, A. G.; Lim, C. Proteins: Struct., Funct., Genet. 1997, 28, 481. (34) d’Auvergne, E. J.; Gooley, P. R. J. Biomol. NMR 2003, 25, 25. (35) Idiyatullin, D.; Daragan, V. A.; Mayo, K. H. J. Magn. Reson. 2003, 161, 118. (36) Choy, W.-Y.; Kay, L. E. J. Biomol. NMR 2003, 25, 325. (37) Vugmeyster, L.; Raleigh, D. P.; Palmer, A. G., III.; Vugmeister, B. E. J. Am. Chem. Soc. 2003, 125, 8400. (38) Perico, A.; Mormino, M.; Urbani, R.; Cesa`ro, A.; Tylianakis, E.; Dais, P.; Brant, D. A. J. Phys. Chem. B 1999, 103, 8162. (39) Xu, Q.; Bush, C. A. Biochemistry 1996, 35, 14512. (40) Martin-Pastor, M.; Bush, C. A. Biopolymers 2000, 54, 235. (41) Henderson, T. J.; Venable, R. M.; Egan, W. J. Am. Chem. Soc. 2003, 125, 2930. (42) Johnson, M. A.; Pinto, B. M. J. Am. Chem. Soc. 2002, 124, 15368. (43) Rosen, J.; Robobi, A.; Nyholm, P.-G. Carbohydr. Res. 2002, 337, 1633. (44) Dabrowski, J.; Kozar, T.; Grosskurth, H.; Nifant’ev, N. E. J. Am. Chem. Soc. 1995, 117, 5534. (45) Landersjo¨, C.; Stenutz, R.; Widmalm, G. J. Am. Chem. Soc. 1997, 119, 8695. (46) Hardy, B. J.; Sarko, A. J. Comput. Chem. 1993, 14, 831. (47) Letardi, S.; La Penna, G.; Chiessi, E.; Perico, A.; Cesa`ro, A. Macromolecules 2002, 35, 286.

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