3281
INTRODUCTION
Most of our knowledge concerning the adaptation of proteins to
varied thermal habitats comes from research on enzymes
(Hochachka and Somero, 2002). According to the corresponding
states theory, differences in the thermal sensitivity of
Michaelis–Menten constants (K
m
) of orthologous enzymes from
poikilotherms adapted to different thermal regimes are driven by
subtle changes in protein primary structure, which in turn lead to
changes in conformational flexibility (Somero, 1978; Somero,
1983). These changes in conformational flexibility offset the
function-altering effects of a change in temperature, thus
conserving optimal function at physiological temperature. In
thermodynamic terms, at lower temperatures less heat/energy is
available to drive physiochemical reactions. Cold-adapted
orthologs respond with a smaller net enthalpy change and a larger
change in entropy than warm/temperate-adapted orthologs, which
compensates for the reduced temperature/kinetic energy. This leads
to a similar change in free energy and conserved binding ability
at physiological temperatures (Somero, 1978; Somero, 1983;
Somero, 1995; Jaenicke, 2000; Hochachka and Somero, 2002).
Over evolutionary time scales, thermal compensation is
accomplished through adjustments in protein primary structure. It
appears that these compensating amino acid substitutions are
generally excluded from the active site residues, which tend to be
conserved to maintain substrate specificity (Wilks et al., 1988;
Golding and Dean, 1998; Fields, 2001).
In prior work, the model derived from enzyme systems was
extended to the non-catalytic Ca
2+
-binding protein parvalbumin
(Erickson et al., 2005; Erickson and Moerland, 2006). Parvalbumins
(PVs) are small (~10–12 kDa), acidic (pI ~3–5) proteins that are
unique to vertebrates and are present at high levels in the cytosol
of fast-twitch skeletal muscle (Rall, 1996). The consensus view of
PV function is that it acts as a soluble Ca
2+
buffer in fast-twitch
muscle cells. By allowing faster unloading of troponin C, the
presence of PV leads to faster contraction/relaxation cycles (Rall,
1996). Isoforms of PV purified from white muscle of cold-adapted
Antarctic fish of the Perciformes sub-order Notothenioidei and from
temperate counterparts displayed thermal sensitivity patterns similar
to those of enzymes of species adapted to different temperatures.
Specifically, at a common measurement temperature Antarctic fish
PVs showed a weaker binding affinity, as evidenced by a higher
Ca
2+
dissociation constant (K
d
), than temperate counterparts, but at
physiological temperatures function was highly conserved (Erickson
et al., 2005; Erickson and Moerland, 2006). A specific structural
mechanism leading to the thermal sensitivity pattern found in
Antarctic fish PVs, however, has yet to be elucidated.
SUMMARY
Parvalbumins (PVs) from Antarctic notothenioid fishes display a pattern of thermal adaptation that likely reflects evolutionary
changes in protein conformational flexibility. We have used ancestral sequence reconstruction and homology modeling to identify
two amino acid changes that could potentially account for the present thermal sensitivity pattern of Antarctic fish PVs compared
with a PV from a theoretical warm-adapted ancestral fish. To test this hypothesis, ancient PVs were resurrected in the lab using
PV from the notothenioid Gobionotothen gibberifrons as a platform for introducing mutations comparable to the reconstructed
ancestral PV sequences. The wild-type PV (WT) as well as three mutant expression constructs were engineered: lysine 8 to
asparagine (K8N), lysine 26 to asparagine (K26N) and a double mutant (DM). Calcium equilibrium dissociation constants (K
d
)
versus temperature curves for all mutants were right-shifted, as predicted, relative to that of WT PV. The K
d
values for the K8N
and K26N single mutants were virtually identical at all temperatures and showed an intermediate level of thermal sensitivity. The
DM construct displayed a full conversion of thermal sensitivity pattern to that of a PV from a warm/temperate-adapted fish.
Additionally, the K
d
versus temperature curve for the WT construct revealed greater thermal sensitivity compared with the mutant
constructs. Measurements of the rates of Ca
2+
dissociation (k
off
) showed that all mutants generally had slower k
off
values than WT
at all temperatures. Calculated rates of Ca
2+
binding (k
on
) for the K8N and K26N mutants were similar to values for the WT PV at
all temperatures. In contrast, the calculated k
on
values for the DM PV were faster, providing mechanistic insights into the nature
of potentially adaptive changes in Ca
2+
binding in this PV. The overall results suggest that the current thermal phenotype of
Antarctic PVs can be recapitulated by just two amino acid substitutions.
Supplementary material available online at http://jeb.biologists.org/cgi/content/full/215/18/3281/DC1
Key words: ancestral sequence reconstruction, EF-hand, environmental adaptation, Notothenioidei, protein structure/function, Southern Ocean.
Received 25 January 2012; Accepted 23 May 2012
The Journal of Experimental Biology 215, 3281-3292
© 2012. Published by The Company of Biologists Ltd
doi:10.1242/jeb.070615
RESEARCH ARTICLE
Resurrecting prehistoric parvalbumins to explore the evolution of thermal
compensation in extant Antarctic fish parvalbumins
A. Carl Whittington
1,
* and Timothy S. Moerland
2
1
Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA and
2
Department of Biological Sciences,
Kent State University, Kent, OH 44242, USA
*Author for correspondence (awhittington@chem.fsu.edu)
THEJOURNALOFEXPERIMENTALBIOLOGY