In search of novel folds : protein evolution via non-homologous recombination : a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Albany, New Zealand
The emergence of proteins from short peptides or subdomains, facilitated
by the duplication and fusion of the minigenes encoding them, is believed to
have played a role in the origin of life. In this study it was hypothesised that
new domains or basic elements of protein structure, may result from nonhomologous
recombination of the genes coding for smaller subdomains.
The hypothesis was tested by randomly recombining two distantly related
(βα)8-barrel proteins: Escherichia coli phosphoribosylanthranilate isomerase
(PRAI), and β subunit of voltage dependent K+ channels (Kvβ2) from Rattus
norvegicus. The aim was to identify new, folded structures, which may or may
not be (βα)8-barrels. Incremental truncation (ITCHY), a method for
fragmenting and randomly recombining genes, was used to mimic in vivo
non-homologous recombination and to create a library of chimeric variants.
Clones from the library were selected for right reading frame and solubility
(foldability) of the recombined chimeras, using the pSALect selection system.
Out of the six clones identified as soluble by pSALect, only one (P25K86) was
found to be actually soluble. The protein, P25K86, was found to form
oligomers and on treatment with a reducing agent, β-mercaptoethanol the
multimeric state disappeared. The protein has three cysteines and one of the
cysteines (Cys56) was found to mediate in the bond formation, thus giving a
dimeric state. An engineered version of P25K86 that has the Cys56 replaced
by serine was expressed as a monomer and additionally it was found to be
As the pSALect folding selection system reported false positives, i.e. only one
of the six chimeras was actually soluble, it was concluded that the in vivo
solubility selection system was leaky. A series of experiments were
conducted so as to improve pSALect that led to the creation of pFoldM – a
more stringent selection system, discussed in chapter 4. Comparing the
newer improved version with the old, two more interesting chimeras were
A total of 240,000 non-homologous recombination events were created in
vitro and three soluble chimeras (evolutionary solutions) were found. Data
from circular dichroism spectroscopy (CD) combined with heteronuclear
single quantum coherence (HSQC) spectra suggest that the proteins,
P24K89 and P25K86, are present in a molten globule state. ITCHY, as a
means of mimicking the subdomain assembly model, was applied in vitro.
The discovery of two interesting chimeras (P25K86 and P24K89) using highthroughput
engineering experiments widens the possibilities of exploring
the protein structure space, and perhaps offers close encounters with these
never born proteins that may be trapped in an ensemble of fluctuating (structured and unstructured) states.