Noninvasive in situ observation of the crystallization kinetics of biological macromolecules by confocal laser scanning microscopy

P. Mühlig, Th Klupsch*, U. Kaulmann, R. Hilgenfeld

*Korrespondierende/r Autor/-in für diese Arbeit
9 Zitate (Scopus)

Abstract

High-resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein crystal growth kinetics. Because the resolution of CLSM is not diffraction-limited by the object, it is possible to visualize, under certain conditions, objects in molecular dimensions. A modified batch technique is applied which allows the growth kinetics of sufficiently small crystallites fixed at the lower side of a cover glass, within a hanging drop, to be studied in reflected light near the total reflection angle. A gap, or cavity, filled with solution is formed between the cover glass and the upper crystal face, which acts to fix small crystallites by hydrodynamic friction forces. The cavity height enables the propagation of molecular steps across the upper crystal face without constraint, so that the propagation velocity and geometrical parameters can be measured by CLSM. The layer growth kinetics of monoclinic crystallites of a long-acting insulin derivative (Insulin Glargine) is investigated. For a twofold supersaturation of the solution, the growth is governed by 2D nucleation at the edges of the crystallites followed by a spreading of molecular steps. The layer growth kinetics are well fitted by the simple cubic kinetic lattice model. We find that only about one of a thousand solute (protein) molecules which push a kink place due to their Brownian motion becomes really incorporated into the growing crystal.

OriginalspracheEnglisch
ZeitschriftJournal of Structural Biology
Jahrgang142
Ausgabenummer1
Seiten (von - bis)47-55
Seitenumfang9
ISSN1047-8477
DOIs
PublikationsstatusVeröffentlicht - 01.04.2003

Strategische Forschungsbereiche und Zentren

  • Forschungsschwerpunkt: Infektion und Entzündung - Zentrum für Infektions- und Entzündungsforschung Lübeck (ZIEL)

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