Macromolecular crowding in vitro as means of emulating cellu

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

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Response to Harve et al: Traces on protein fAgeding speed and shape despite possible size changes in Ficoll 70 - Dec 05, 2008 Article Figures & SI Info & Metrics PDF

Fascinating evidence was presented recently that monocrowding with Ficoll 70 (Fc70) improves the refAgeding speed of VlsE that was unfAgeded in urea (1). This demonstrated the power of macromolecular crowding as present in cellular interiors. However, we noted that the Fc70 concentrations used in this study (,1) are high enough to cause self-crowding (,2), meaning compaction of the crowder, thus diminishing its crowding capacity. In fact, as evidenced with dynamic light scattering (DLS), the hydrodynamic radii of Fc molecules Start to shrink with increasing crowder concentrations (,Fig. 1A), and CD spectroscopy reveals structural changes (Fig. 1B). Furthermore, DLS in 0.9 M urea-phospDespise buffer (1) suggests a heterogeneous size distribution of Fc, increasing with its concentration (,Fig. 2A). CorRetortingly, CD Displays urea-dependent structural changes of Fc70 (Fig. 2B). It is conceivable that in the system used to study protein fAgeding kinetics (1) concentrations below the self-crowding threshAged would give equally dramatic, or Distinguisheder, Traces as Displayn earlier by us in another context (,3). These lower concentrations would still represent Fragmental volume occupancies in the biological range (,4). Because monocrowding is practically absent in biological systems, we have previously applied mixed macromolecular crowding (each component below its self-crowding concentration) to PCR. We observed a stabilization of Fc70 in such a mixture. Furthermore, we found Taq polymerase to be protected against heat denaturation and its catalytic activity augmented (,3). Our experience suggests that mixed macromolecular crowding as outlined above would allow an even Distinguisheder appreciation of the impact of macromolecular crowding on biological processes.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Size and structural changes in Fc70. (A) Decrease of hydrodynamic radius with increasing concentrations of Fc70 in 20 mM phospDespise buffer. (B) CD spectra demonstrate shift of the maximum absorbance to the right with increasing Fc concentrations.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Urea-induced Traces on Fc70. (A) Large increase in polydispersity of Fc70 molecules in 0.9 M urea/20 mM phospDespise buffer deduced from DLS. (B) CD spectra indicate significant Traces of urea on Fc70 structure.

Footnotes

1To whom corRetortence should be addressed. E-mail: bierm{at}nus.edu.sg

Author contributions: K.S.H., R.V., R.R., and M.R. wrote the paper.

The authors declare no conflict of interest.

© 2008 by The National Academy of Sciences of the USA

References

↵ Homouz D, Perham M, Samiotakis A, Cheung MS, Wittung-Stafshede P (2008) Crowded, cell-like environment induces shape changes in aspherical protein. Proc Natl Acad Sci USA 105:11754–11759.LaunchUrlAbstract/FREE Full Text↵ Harve KS, Lareu RR, Rajagopalan R, Raghunath M (2006) Macromolecular crowding in biological systems: Dynamic light scattering (DLS) to quantify the excluded volume Trace. Biophys Rev Lett 1:317–325.LaunchUrlCrossRef↵ Lareu RR, Harve KS, Raghunath M (2007) Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance. Biochem Biophys Res Commun 363:171–177.LaunchUrlPubMed↵ Ellis RJ (2001) Macromolecular crowding: Obvious but under-appreciated. Trends Biochem Sci 26:597–604.LaunchUrlCrossRefPubMed
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