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PEDS Advance Access originally published online on February 20, 2008
Protein Engineering Design and Selection 2008 21(5):303-310; doi:10.1093/protein/gzn005
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Thermostability promotes the cooperative function of split adenylate kinases

Peter Q. Nguyen, Shirley Liu, Jeremy C. Thompson and Jonathan J. Silberg1

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA

1 To whom correspondence should be addressed. E-mail: joff{at}rice.edu

Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AKBs) and the hyperthermophile Thermotoga neapolitana (AKTn). Complementation studies revealed that the split AKTn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AKBs. However, weak complementation occurred when the AKBs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AKTn had higher levels of zinc, greater secondary structure, and >103-fold more activity than the AKBs pair, albeit 17-fold less active than full-length AKTn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.

Keywords: adenylate kinase/hyperthermophile/protein design/protein fragment complementation/protein thermostability

Received November 14, 2007; revised January 12, 2008; accepted January 18, 2008.

Abbreviations: AKBs, B. subtilis AK; AKEc, E. coli AK; AKTn, T. neapolitana AK; CD, circular dichroism; DTT, dithiothreitol; LB, luria broth; MMTS, methyl methanethiolsulfonate; PAR, 4-(2-pyridylazo) resorcinol; Tm, midpoint of thermal denaturation.


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