Creatinase: Using Increased Entropy to Improve the Activity and Thermostability

Abstract

Improving protein thermostability in mutagenesis-based enzyme engineering was often achieved by enhancing interresidue interactions via mutation to increase the enthalpy penalty of unfolding. However, this approach may trade off the functional activity due to the loss of structural flexibility of the biomolecule. Here, by performing X-ray crystallography, enzymatic kinetic experiments, neutron scattering, and thermodynamical measurements, we compared the structures, catalytic behaviors, dynamics, and thermostability between a wild-type creatinase and its four-point mutant. We found that the mutant is an entropy-driven thermostable protein with higher structural flexibility, i.e., higher conformational entropy, in the folded state compared to the wild type. The increased conformational entropy of the mutant in the folded state can reduce the entropy gain during unfolding and thus renders it greater thermostability. Moreover, the increased structural flexibility, particularly around the catalytic site, can broaden the mutant's working temperature range and considerably improve its activity at ambient conditions, which is crucial for its application in diagnosing kidney diseases. Complementary all-atom molecular dynamics simulations indicated that the four mutations replaced several of the strong interresidue interactions (electrostatic interactions and hydrogen bonds) with weak hydrophobic interactions. These substitutions not only release the structural flexibility to promote the thermostability and enzymatic activity of the protein but they also preserve the protein structure from collapsing. Our findings may pave a route for the entropy-driven strategy to design proteins with high thermostability and activity.

Making sense of chaos: uncovering the mechanisms of conformational entropy

“…In Adenylate kinase, mutations that affect local unfolding, as measured by differential scanning calorimetry and NMR, lead to favorable catalytic properties through entropic stabilization 64 . Neutron scattering, which reveals average changes in flexibility, suggested that an engineered creatinase is stablized, relative to wildtype, by increased entropy in the folded state 65 .…”

A Bibliometric Analysis of Scholarly Publication on Protein Folding From 2018 to 2022

“…Inactive protein functions raise various diseases like Huntington's disease (8), Alzheimer's disease (12), Cystic fibrosis (13), Parkinson's disease ( 14), Gaucher's disease followed by allergies (15). Chaperon helps to keep the protein on the right path if the protein debate from the holding pattern.Now a days,various experimental techniques are used for protein folding such as Circular dichroism (16), X-ray crystallography, Fluorescence spectroscopy,X-ray crystallography etc. (17); However, Kubelka et al, reported that the time scale of the protein folding depends on its size and topology (18).…”

Semantical and Geometrical Protein Encoding Toward Enhanced Bioactivity and Thermostability

“…While wild-type proteins exhibit optimal bio-functionality in their native environments, industrial applications often demand adaptation to conditions such as high temperature, high pressure, strong acidity, and strong alkalinity. The constrained efficacy of proteins in meeting the stringent requirements of industrial functioning environments hinders their widespread applications [2], [18], [19], [68], [78]. Consequently, there arises a need to engineer these natural proteins to enhance their functionalities, aligning them with the demands of both industrial and scientific applications.…”