Enhancing the thermal stability of a single-chain Fv fragment by in vivo global fluorination of the proline residues

Single-chain Fv (scFv) format protein is a commonly used analytical tool for diagnostic and therapeutic applications. The usage of scFv antibody fragments in therapeutic applications has demonstrated that they need to have high thermostability. Many rational or irrational methods have been described erstwhile to engineer or improve the stability of scFv proteins by modifications of natural amino acid. Here we have demonstrated an alternate strategy to efficiently improve the thermostability of scFvs by non-canonical amino acid. Previously, fluoroprolines have been proven as a choice to tune the stability of many polypeptides and few globular proteins. Hence we exploited the usage of fluoroproline to enhance the thermal stability of scFv by replacing the natural proline on the framework regions of scFv that influence the folding or stability. To demonstrate our approach, a bacterial cytoplasmic foldable and humanized anti-c-Met scFv (hu-MscFv) was used. The hu-MscFv proline sites were successfully incorporated with (2S,4R)-4-fluoroproline without affecting its structure and function by the in vivo residue specific global replacement method which exploits bacterial auxotrophic system. The time-dependent temperature effect on the activity of fluorinated hu-MscFv revealed its increased stability at 40 °C along with improved half-life than the hu-MscFv with natural proline. Further model based structure analysis on hu-MscFv with fluoroproline indicated that the fluorine atoms were able to establish new favourable dipole interactions with neighbouring polar groups in their local micro environments that rationalizes its improved thermostability. Moreover the scFv sequence based statistical analysis strongly supports the fact that this method can be applied to any target scFv, since they contain high frequency conserved proline sites in their framework regions

A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

<div><p>Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.</p> </div

Comparison of salt-bridge and hydrogen bonds interactions of the GFP variants.

‘+’<p>– indicates the presence of mutation of lysine to arginine.</p>‘−’<p>– indicates the absence of mutation of lysine to arginine.</p><p>‘*’– new interactions.</p

Computational screening of potential non-immunoglobulin scaffolds using overlapped conserved residues (OCR)-based fingerprints

Cystatins and lipocalins have attracted considerable interest for their potential applications in non-immunoglobulin protein scaffold engineering. In the present study, their potential homologs were screened computationally from non-redundant protein sequence database based on the overlapped conserved residues (OCR)-fingerprints, which can detect the protein family with low sequence identity, such as cystatins and lipocalins. Two types of OCR-fingerprints for each family were designed and showed very high detection efficiency (>90%). The protein sequence database was scanned by the fingerprints, which yielded the hypothetical sequences for cystatins and lipocalins. The hypothetical sequences were validated further based on their sequence motifs and structural models, which allowed an identification of the potential homologs of cystatins and lipocalins

Effect of ionic detergents on the stability of the GFP variants.

<p>Protein samples were incubated with 1% SDS (A), 1% SDBS (B), 1% CTAB (C) and 1% DTAC (D) at 50°C for different time intervals and the remaining fluorescence was measured. The fluorescence at time zero at the respective detergent was taken into 100%. (Error bar – Standard deviation of the three independent experiments).</p

Salt-bridge and hydrogen bond interactions associated with the lysine and arginine residues in GFP.

*<p>- three dimensional structure information is not known.</p

Effect of temperature on the stability of the GFP variants.

<p>A) Protein samples were incubated at different temperatures for 30 minutes and the remaining fluorescence was measured. The fluorescence at time zero at the respective temperatures was taken into 100%. B) Protein samples were incubated at 70°C for different time intervals and the remaining fluorescence was measured. The fluorescence at time zero was taken into 100%. (Error bar – Standard deviation of the three independent experiments).</p

Effect of pH on the stability of the GFP variants.

<p>A) Protein samples were incubated at 60°C for 30 minutes in different pH buffer solutions and the remaining fluorescence was measured. The fluorescence at time zero at the respective buffer was taken into 100%. B) Protein samples in KCl buffer with pH 13.0 were incubated at 60°C for different time intervals and the remaining fluorescence was measured. The fluorescence at time zero was taken into 100%. (Error bar – Standard deviation of the three independent experiments).</p

Spectral properties of the GFP variants.

<p>A) Excitation and B) Emission spectrum of the GFPcon and GFP14R. All the amplitudes were arbitrarily normalized to a maximum value of 1.0. (au – arbitrary units).</p