Most proteins contain small cavities that can be filled by replacing cavity-lining residues by larger ones. Since shortening mutations in hydrophobic cores tend to destabilize proteins, it is expected that cavity-filling mutations may conversely increase protein stability. We have filled three small cavities in apoflavodoxin and determined by NMR and equilibrium unfolding analysis their impact in protein structure and stability. The smallest cavity (14 A3) has been filled, at two different positions, with a variety of residues and, in all cases, the mutant proteins are locally unfolded, their structure and energetics resembling those of an equilibrium intermediate of the thermal unfolding of the wild-type protein. In contrast, two slightly larger cavities of 20 A3 and 21 A3 have been filled with Val to Ile or Val to Leu mutations and the mutants preserve both the native fold and the equilibrium unfolding mechanism. From the known relationship, observed in shortening mutations, between stability changes and the differential hydrophobicity of the exchanged residues and the volume of the cavities, the filling of these apoflavodoxin cavities is expected to stabilize the protein by approximately 1.5 kcal mol(-1). However, both urea and thermal denaturation analysis reveal much more modest stabilizations, ranging from 0.0 kcal mol(-1) to 0.6 kcal mol(-1), which reflects that the accommodation of single extra methyl groups in small cavities requires some rearrangement, necessarily destabilizing, that lowers the expected theoretical stabilization. As the size of these cavities is representative of that of the typical small, empty cavities found in most proteins, it seems unlikely that filling this type of cavities will give rise to large stabilizations. Study holds ProTherm entries: 22077, 22078, 22079, 22080, 22081, 22082, 22083, 22084, 22085, 22086, 22087, 22088, 22089, 22090, 22091, 22092, 22093, 22094, 22095, 22096, 22097, 22098, 22099, 22100, 22101, 22102, 22103, 22104, 22105, 22106, 22107, 22108, 22109, 22110, 22111, 22112 Extra Details: apo form; Native to Intermediate; Global fitting of the four thermal unfolding curves recorded for each mutant protein (fluorescencel, far-UV CD, near-UV CD, and near-UV Abs). protein cavity; protein stability; protein intermediate; protein folding; van der Waals
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:53 p.m.
|Number of data points||77|
|Proteins||Flavodoxin ; Flavodoxin|
|Assays/Quantities/Protocols||Experimental Assay: ddG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: ddG ; Experimental Assay: dTm units:°C ; Experimental Assay: dTm units:K ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence SKKIGLFYGTQTGKTESVAEIIRDEFGNDVVTLHDVSQAEVTDLNDYQYLIIGCPTWNIGELQSDWEGLYSELDDVDFNGKLVAYFGTGDQIGYADNFQDAIGILEEKISQRGGKTVGYWSTDGYDFNDSKALRNGKFVGLALDEDNQSDLTDDRIKSWVAQLKSEFGL|