Nuclear magnetic resonance relaxation studies of poly(hydroxybutyrate) in whole cells and in artificial granules Graeme L. Shaw, Melissa K. Melby, Daniel M. Horowitz, James Keeler and Jeremy K. M. Sanders* Cambridge Centre for Molecular Recognition, University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, UK Received 6 September 1993; revised 22 October 1993 The physical state of poly(hydroxybutyrate) (PH [3) in whole cells and in the form of artificial • 13 biomimetic granules has been probed using C nuclear magnetic resonance (NMR) spectroscopy. Studies on varying concentrations of whole cells of Alcaligenes eutrophus show that changes in the line widths of PHB in whole cells do not correlate with changes in transverse relaxation times. Solid-state magic-angle spinning NMR studies demonstrate that the line broadening results from a reduction in the static field homogeneity rather than from intrinsic properties of the PHB within the cells. Transverse and longitudinal relaxation times of PHB in whole cells and in artificial granules are similar, indicating similarities in structure and mobility. Keywords: granules; nuclear magnetic resonance; poly(hydroxybutyrate) Poly(3-hydroxybutyrate) (PHB, Figure 1) is an optically active polyester that is synthesized by a wide variety of bacteria 1'2. The isolated polymer is highly crystalline and has generated great interest due to its industrial applications as a biodegradable thermoplastic. The polymer functions as a carbon and energy storage compound in times of nutrient limitation in the cell, where it is found in discrete granules• Early studies on isolated granules 3'4 incorrectly led to the conclusion that native granules must contain crystalline polymer, but, in the late 1980s, we and others demonstrated that the polymer is an amorphous elastomer within the cell 5-7. This discovery was based primarily on the observation of narrow ~aC nuclear magnetic resonance (NMR) lines for PHB in vivo using solution techniques, and on the absence of crystalline peaks in wide-angle X-ray scattering studies. The realization that PHB is a mobile elastomer in vivo also provided an explanation for the fact that it is rapidly accessible to the action of intracellular depolymerases; this had been difficult to rationalize for a crystalline solid. More recently, observations on the cells and granules after treatments such as freeze-drying and isolation procedures led to the proposal of a four-state model for the PHB granule s. The existence of a 'relatively mobile state' was proposed on the basis of reversible increases in line broadening in the 13C NMR spectra on freeze-drying. This was interpreted as indicative of a partial loss of molecular mobility. While loss of molecular mobility may indeed cause line broadening, the reverse conclusion, i.e. that line broadening indicates loss of *To whomcorrespondenceshouldbe addressed molecular mobility, is not necessarily true. Furthermore, the original conclusion that a plasticizer was responsible for molecular mobility has been challenged by a new kinetic model 9'1°, and by our resulting preparation of artificial biomimetic granules lz. We report here the use of high-resolution solution- state and solid-state ~ 3C NMR spectroscopy to investigate the phenomenon of line broadening in increasing cell concentrations. The dependence of transverse relaxation times on cell concentration has been measured, including a sample of freeze-dried cells• Transverse and longitudinal relaxation times have also been measured for native and artificial granules. We conclude that there is no loss of mobility of PHB within the cells on freeze-drying. Experimental Alcaligenes eutrophus tron (a naturally selected high- yielding strain supplied by Zeneca Biopolymers, Billingham, UK) was grown on minimal salts medium Figure 1 Me 0 Poly(3-hydroxybutyrate) 0141-8130/94/020059-05 © 1994Butterworth-Heinemarm Limited Int. J. Biol. Macromol. Volume 16 Number 2 1994 59