Related Experiment Video
Updated: Aug 8, 2026
11:28
Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
Published on: March 31, 2012
Design of fully active FGF-1 variants with increased stability
Malgorzata Zakrzewska1, Daniel Krowarsch, Antoni Wiedlocha
1Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland.
Protein Engineering, Design & Selection : PEDS
|October 8, 2004
Summary
Researchers enhanced fibroblast growth factor 1 (FGF-1) stability using a homology approach. Mutants showed increased thermodynamic stability without compromising essential biological activities, offering therapeutic potential.
Area of Science:
- Biochemistry
- Protein Engineering
- Molecular Biology
Background:
- Fibroblast growth factor 1 (FGF-1) is a key mitogen involved in crucial biological processes like morphogenesis, angiogenesis, and wound healing.
- Its therapeutic potential is limited by its inherent instability.
- Improving FGF-1 stability is critical for its successful application in medicine.
Purpose of the Study:
- To engineer FGF-1 mutants with enhanced thermodynamic stability.
- To assess the impact of mutations on FGF-1's biological functions.
- To establish a method for improving protein stability without functional loss.
Main Methods:
- Utilized homologous sequence and structure comparisons to design FGF-1 mutants.
- Constructed 16 FGF-1 mutants.
- Determined thermodynamic stability using chemical and heat denaturation assays.
- Evaluated biological activities including DNA synthesis stimulation, MAP kinase activation, and FGF receptor binding.
Main Results:
- Engineered FGF-1 mutants exhibited increased thermodynamic stability, with some up to 7.8°C more stable than wild-type.
- Additive effects on stability were observed in multiple mutants.
- No introduced mutations impaired FGF-1's biological activities, including receptor binding and downstream signaling.
- Homology-based protein engineering effectively enhanced stability without compromising function.
Conclusions:
- The homology approach is effective for improving protein thermodynamic stability.
- Engineered FGF-1 mutants offer enhanced stability for therapeutic applications.
- This strategy provides a foundation for developing more stable protein therapeutics.
Related Concept Videos
01:49Conserved Binding Sites
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
00:49Ligand Binding and Linkage
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
01:57Protein Complexes with Interchangeable Parts
Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
01:22Activation and Inactivation of G Proteins
Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high affinity and are together...