Life exists over a wide temperature range, from as low as -15°C to as high as 122°C . On the upper end of the temperature spectrum, thermophiles and hyperthermophiles have been studied extensively by the scientific community, particularly the molecular mechanisms that support protein structure and function at high temperatures. For example, compact and strong hydrophobic packing is typically found in most cores of thermophilic proteins, which increases the energy needed to unfold the protein, making it possible for thermophilic proteins to retain native structure at high temperatures . Indeed, a strong correlation exists between high core packing density and thermostability [2–4]. We are also intrigued by the obverse - organisms that survive optimally at cold temperatures; harsh environments with restricted molecular mobility and reduced reaction kinetics that hinder myriad cellular and biomolecular processes [5–7].
Psychrophiles, "cold-loving" microorganisms, have adapted to life at low temperatures by using a variety of mechanisms. These include the production of anti-freeze and cold-shock proteins, alterations in membrane composition, and overexpression of proteins that destabilize DNA structures, among other mechanisms [8–10]. Some of the more interesting adaptations are found in psychrophilic enzymes. In particular, psychrophilic enzymes typically have a higher occurrence of nonpolar residues on their surface, which destabilizes water structure around the enzyme such that water properties are considerably distinct from bulk water, and a lower abundance of arginine and proline residues, which increases backbone flexibility. Moreover, fewer weak interactions, such as hydrogen bonds, reduce the degree of packing within the enzyme core and make psychrophilic enzymes more conformationally flexible [[11–13]; and references therein]. Increased flexibility has been proposed as a major molecular mechanism for the evolution of cold adapted enzymes, a hypothesis supported by spectroscopic analysis (e.g., nuclear magnetic resonance , dynamic fluorescence quenching  and molecular dynamics (MD) simulations ).
Psychrophilic enzymes have been shown to lose more entropy upon transition state activation than their mesophilic counterparts, suggesting that the psychrophilic proteins exist in a more disordered ground state [17, 9]. This intuitively makes sense, since in order for psychrophilic enzymes to be more active at low temperatures they must have more kinetic energy . Nonetheless, the structural basis of this flexibility remains unclear. For example, an MD simulations study of one-on-one comparisons of trypsin from Atlantic salmon and its mesophilic counterpart  found that the psychrophilic trypsin possesses higher flexibility close to the active site parts. Interestingly, the active site is where there is the most significant difference in amino acid composition between mesophilic and psychrophilic trypsins. However, other psychrophilic enzymes exhibit regions of flexibility in places distant from the active site. For example, psychrophilic uracil-DNA glycosylases  have fewer strong ion-pair interactions near the C-terminus than their mesophilic counterparts. This local difference results in increased flexibility in the psychrophilic enzymes. Psychrophilic alpha-amylases  show greater overall flexibility than the mesophilic/thermophilic counterparts. This is consistent with thermal unfolding experiments, where the psychrophilic enzymes unfold at low temperatures cooperatively without intermediates due to fewer stabilizing interactions, while thermophilic amylases show intermediates during unfolding, indicating that there are regions with greater rigidity than others. The different strategies that psychrophilic enzymes use to adapt to colder environments have resulted in a number of divergent viewpoints on the influence of local and global protein flexibility on cold adaptation.
An interesting structural property of proteins, particularly as it relates to thermostability, is cavity number and size. Protein thermostability appears to diminish when cavities are created in the protein . Such cavities represent packing defects in the protein core. In the case of psychrophilic proteins, only a few studies have addressed these cavities, and then only with a small set of proteins [11, 22]. No clear relationship was observed between psychrophily and total void-volume in the enzyme core, as might be expected from the strong hydrophobic cores present in thermophilic proteins. However, an increased number of three-dimensional structures of psychrophilic proteins have become available and offer the opportunity to revisit such a structural analysis. Here we test the hypothesis that cavity properties in psychrophilic enzymes endow these proteins with the increased conformationally flexibility necessary to function optimally at low temperatures.
In the current work we investigated differences in the average cavity volume and crystallographic waters between psychrophilic and mesophilic homologs. A non-redundant set of 20 psychrophilic enzymes was examined, with each paired to a homologous mesophilic enzyme (sequence identity above 35% to 76%), and all with high resolution crystal structures. In addition to counting cavities and calculating void volumes, we evaluated amino acid frequencies in residue positions surrounding cavities. We present evidence that the average cavity size of psychrophilic enzymes is larger, and contains more surrounding hydrophilic groups, than their mesophilic counterparts. These findings support a hypothesis that psychrophilic enzymes may have a predisposition to having more water molecules within their cavities, which consequently increases enzyme conformational dynamics, leading to greater activity under the rigidifying cold environment. The results provide a potential strategy by which optimal temperature could be altered.