Materials and Methods

4,2,1 Sam ple Preparation

Galpl-4[U-^^C, ^HJGlc was prepared by chemoenzymatic synthesis performed in a

2.5 ml solution containing lOmM UDP-galactose and lOmM U -‘^C, glucose (from

M artek Biosciences) in 50mM phosphate buffer (pH 7.4). One unit of

galactosyltransferase was added with ImM lactalbumin and Mn^"^ to a concentration

of 5mM, and the mixture was incubated at 37"C overnight.

U-^^C“G alpl-4G lc was prepared by chemoenzymatic synthesis performed in 2.5 ml

solution of 50mM Tris (pH 7.8), 5mM M gCh, 50mM ATP and 50mM Galactose.

One unit of Galactokinase was added and the mixture was incubated at 37"C for 24

hours. To this was added 1 unit of Uridylyl transferase and lOmM UDP-glucose, this

was incubated at 37"C for 24 hours. After this time 50mM glucose, 1 unit of

galactosyltransferase and ImM lactalbumin was added. The solution was left for a

further 24 hours at 37‘’C.

[*^C]Gaipi-4[“H,^'^C]Glc was prepared in the same way as above, except in the final

stage 50mM 'H ,] glucose was added instead of glucose.

The disaccharide products were purified by Biogel P-2 column (2.5 cm * 100cm) chromatography, with H2O eluant.

Samples for experiments at 30“C were dissolved and lyophUised into 99.96% D2O

three times followed by dissolution into 700|iL D%0. For the observation of the

exchangeable protons samples were dissolved in 750pL of H2O containing 15%

99.96% Acetone-r4 (Cambridge Isotopes). The pH was adjusted to -5.7 by careful

step-wise addition of dilute HCl or NaOH and transferred to a 5mm NMR tube. The

sample was degassed by sonication for about one minute.

4,2,2 N M R Experim ents

NMR spectra were obtained at 300K with a ^H reference frequency of 500.12M Hz

on a Variai! Unity^ spectrometer equipped with a self shielded z gradient triple

resonance probe. All spectra were recorded in the phase-sensitive mode with use of

the States (States et al., 1982) method for quadrature detection.

Two-dimensional {^^C}^H heteronuclear Overhauser effect (HOESY) experiments

were recorded on G aipi-4[U -‘‘^C, “H]Glc as described [chapter 2].

Two-dimensional homonuclear ^H ROES Y experiments for the measurement of

ROEs to and from exchangeable hydroxyl protons were recorded on G alpl-4G lc at

258 K in HjO/acetone solution (85:15 v/v) with a conventional ROESY pulse

sequence appended with a double pulsed-field gradient echo sequence. Spectra were

acquired with spectral widths of 3600 Hz in each dimension, 1024 complex points in

t2, 256 complex points in t| and with 16 transients per ti increment. The spin-lock

Chapter 4: Solution structure o f Lactose 110

shifted to low-field of the spectrum for the duration of the spin-lock. Prior to Fourier

transformation time-domain data were apodised with cosine-bell functions in each

dimension. Hydroxyl exchange rates were measured using the method described by

Adams and Lerner (Adams, 1992) incorporating a double pulsed-field gradient echo sequence (Hwang, 1995).

Three-dimensional NOESY-HSQC experiments were recorded with spectral widths

of llOOHz in the ti and ti (^H) dinaensions, 6000Hz in the ta dimension, and 128, 32,

and 512 complex points in the ti, ta and ta dimensions, respectively. A mixing time of

500 ms was used. Prior to Fourier transformation, each dimension was apodised with

cosine-bell weighting functions followed by zero filling to 256, 64, and 1024 complex

points, respectively.

Two-dimensional gradient-enhanced long range carbon-carbon correlation (LRCC)

experiments (Bax et al. 1992) were recorded on U -‘^C-Galpl-4Glc with a proton sweep width of 1.1 kHz, a sweep-width of 6.5kHz, and 1024 complex points in ta

and 256 complex points in ti. A total of 64 scans were acquiied per ti increment. *^C-

couphngs evolve and are re-focussed during the delay (2T) of 22.2ms. The values

of the long range coupling constants are derived from the ratios of cross-peaks

Residual dipolar couplings were measured in U-^^C-Gaipi-4Glc in D2O

containing 33% w/v o f a mixture (1:2.9) of diliexanoyl phosphatidylcholine and

dimyristoylphosphatidylcholine as described in chapter 3.

4.2,3 M olecular M odelling

Dynamical simulated annealing calculations and molecular dynamics (MD)

simulations were computed in vacuo as described (Homans & Forster, 1992; Rutherford & Homans, 1994). Random structures were generated by dynamical

quenching. An initial structure was built with pyranose rings in the '‘Ci chaii*

conformation with trial values of phi (tp) and psi (\|/), and subjected to 200ps of

unrestrained molecular dynamics at 750K. A random structure was saved every lOps.

Energy minimisation by restrained simulated annealing was achieved as follows:

Models were equilibrated for lOps with a thermal bath at temperatures 500K, 45OK, 350K, 300K, and then successively for Ips in decreasing steps of lOK, followed by a

further Ips at 5K. The system was minimised using a steepest descents algorithm

until the maximum derivative was less than 0.04 K J mol'^ Â ''. Restraints were

applied as a biharmonic function. NOE contacts were arbitrarily assigned as strong

(1.8Â - 2.7À), medium (1.8Â - 3.3Â), and weak (1.8Â - 5.0Â). Residual dipolar

restraints were incorporated into the molecular dynamics simulation as described

(Homans, 1992; Clore, 1998), using the program XPLOR modified to incorporate

dipolar restraints. Values for the axial and rhombic components of the aUgnment

Chapter 4: Solution structure o f Lactose 112

The MD simulations incorporated homonuclear *H NOE restraints, including those

to and from hydroxyl protons, in the form of time-averaged restraints as described by

Torda (Torda, 1989 and 1990), with a memory time x of 5ps. Theoretical NOE

intensities and spin-coupling constants were computed from molecular dynamics

simulations using in-house written software. Computation of NOE intensities

incorporated a full relaxation matrix approach including a formalism appropriate for

the computation of NOE and ROE data due to fluctuating inter-nuclear distances

arising from internal motions which are fast with respect to the rate of molecular

tumbhng (Tropp, 1980; Homans and Forster, 1992). A single overall isotropic

correlation time for the molecule was assumed, and was obtained by fitting the ratio

of the theoretical diagonal peak to cross-peak intensities for a known, fixed distance

(intra-residue NOE) to the experimentally measured values.