Intrinsic Disorder-Based Design of Stable Globular Proteins

Abstract

Directed stabilization of globular proteins via substitution of a minimal number of amino acid residues is one of the most complicated experimental tasks. This work summarizes our research on the effect of amino acid substitutions on the protein stability utilizing the outputs of the analysis of intrinsic disorder predisposition of target proteins. This allowed us to formulate the basis of one of the possible approaches to the stabilization of globular proteins. The idea is quite simple. To stabilize a protein as a whole, one needs to find its "weakest spot" and stabilize it, but the question is how this weak spot can be found in a query protein. Our approach is based on the utilization of the computational tools for the per-residue evaluation of intrinsic disorder predisposition to search for the "weakest spot" of a query protein (i.e., the region(s) with the highest local predisposition for intrinsic disorder). When such "weakest spot" is found, it can be stabilized through a limited number of point mutations by introducing order-promoting residues at hot spots, thereby increasing structural stability of a protein as a whole. Using this approach, we were able to obtain stable mutant forms of several globular proteins, such as Gαo, GFP, ribosome protein L1, and circular permutant of apical domain of GroEL.

Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy

“…To identify the regions within the luciferase structures which could demonstrate less conformational stability, we used algorithms predicting intrinsically disordered regions relative to the approach described in [ 32 ]. This approach is based on the utilization of computational tools for the per-residue evaluation of the intrinsic disorder predisposition to search for the “weakest spot” of a query protein.…”

“…In the crystal structure of the luciferase [ 13 ], the α-helixes and β-strands were resolved in this region. The apparent contradiction could mean that this part of the subunit requires interaction with the remaining rigidly packed part of the protein to obtain the correct three-dimensional structure [ 32 ]. Thus, our analysis indicates that the observed lower stability of the secondary structure of the V. harveyi luciferase in comparison with the P. leiognathi enzyme could be attributed to the difference in the sequence of the C-terminal part of the β-subunit.…”

A Thermodynamic Atlas of Proteomes Reveals Energetic Innovation across the Tree of Life

“…5 C )? Possibilities for this observation include; 1) the multidomain structure of many eukaryotic proteins, where locally stable domains are interspersed with disordered stretches, such that the average location in PC space reflects both properties; or 2) increased eukaryotic use of mechanisms to stabilize protein structure that do not rely on hydrophobicity, such as hydrogen bonds ( Myers and Pace 1996 ; Pace et al 2014 ), salt bridges ( Bosshard et al 2004 ), conformational entropy ( Matthews et al 1987 ; Pace et al 1988 ; Nagibina et al 2019 ), or covalent linkages ( Fass 2012 ; Wensien et al 2021 ). Related to the second point, the longer average lengths of eukaryotic proteins ( Brocchieri and Karlin 2005 ) increase the temperature dependence of stability for globular proteins with a hydrophobic core, due to the curvature of the free energy of stability as a function of temperature that results from the larger heat capacity change ΔCp of unfolding larger proteins ( Alexander et al 1992 ; Robertson and Murphy 1997 ).…”

In Silico Modeling of the Influence of Environment on Amyloid Folding Using FOD-M Model

“…In the context of the fuzzy oil drop model, the widely discussed role of intrinsically disordered proteins (IDP) in amyloid transformation does not appear to play a critical role, due to the local nature of chain fragments, while amyloid transformation is global [ 129 , 130 , 131 , 132 , 133 , 134 ]. For the analysis of the amyloid transformation based on the fuzzy oil drop model, the characterization of the down-hill and fast-folding protein structures is important because these examples confirm the validity of the assumptions of this model [ 135 , 136 , 137 , 138 , 139 ] (see also Supplementary Materials , S5, Miscellaneous proteins).…”