Abstract
Cold shock proteins (Csp) are small acidic proteins that fold into β-barrel
structures with five anti-parallel β-strands and are involved in essential
cellular processes. Upon temperature downshift the synthesis of Csp proteins
is drastically increased to enable cells to restore growth in the cold. These
proteins facilitate transcription and translation at low temperature by
functioning as RNA chaperones. Csp proteins have been most extensively
studied in Bacteria but very few Csp homologues have been identified and
studied in Archaea. This is the first study examining structural, functional
and biophysical properties of Csp from the Antarctic archaeon Methanogenium
frigidum. The fastidious growth requirements of M. frigidum make it difficult
to cultivate, therefore recombinant methods have been developed for the
expression and characterization of the protein. The analysis by transverse
urea gradient gel electrophoresis (TUG-GE) revealed that M. frigidum Csp
folds by a reversible two-state mechanism and has a low conformational
stability. The spectroscopic analysis of the protein performed by Circular
Dichroism (CD) spectroscopy disclosed features typical of other homologous
proteins. A possible association between Csp and RNA has been proposed
according to MALDI-TOF mass spectrometry analysis. The effect of a Nterminal
polyhistidine affinity tag on the biophysical properties of Csp was
also examined. The biological activity of Csp was investigated by
complementation of an E. coli cold sensitive mutant. These studies revealed
that the M. frigidum Csp is biologically active and can function in E. coli.