Related Experiment Video
Updated: Aug 13, 2026
11:55
In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae
Published on: October 10, 2014
Unveiling a hidden folding intermediate in c-type cytochromes by protein engineering
Alessandro Borgia1, Daniele Bonivento, Carlo Travaglini-Allocatelli
1Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche, Università di Roma "La Sapienza," P.le A. Moro 5, 00185 Rome, Italy.
The Journal of Biological Chemistry
|February 3, 2006
Summary
Researchers engineered a Pseudomonas aeruginosa cytochrome c(551) (Pa cyt c(551)) mutant to stabilize a folding intermediate. This study reveals how protein engineering and structural analysis uncover folding mechanisms and intermediate stability determinants.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Folding Dynamics
Background:
- Protein folding mechanisms are often linked to topology and involve partially structured states.
- Cytochrome c (cyt c) family members are hypothesized to share common folding pathways.
- Pseudomonas aeruginosa cyt c(551) (Pa cyt c(551)) typically exhibits a two-state folding mechanism.
Purpose of the Study:
- To investigate the possibility of altering the folding mechanism of Pa cyt c(551) from two-state to three-state.
- To stabilize a high-energy folding intermediate using rational mutagenesis.
- To elucidate the structural basis for intermediate stabilization.
Main Methods:
- Rational mutagenesis to create a single-site mutant (Phe7 to Ala).
- Kinetic analysis of protein refolding and unfolding.
- X-ray crystallography to determine the structure of the mutant protein.
Main Results:
- The Phe7 to Ala mutant exhibited an altered folding mechanism, with an additional refolding phase and faster unfolding, indicating intermediate stabilization.
- Kinetic data suggested the intermediate is a non-obligatory on-pathway species.
- Crystallographic analysis revealed an internal cavity and altered N-terminal helix in the mutant, providing structural insights into intermediate stabilization.
Conclusions:
- Protein engineering can successfully switch the folding mechanism of Pa cyt c(551) and stabilize a folding intermediate.
- Specific structural features, such as internal cavities and helix length, are critical determinants of intermediate stability.
- The study demonstrates the power of integrating kinetics, crystallography, and mutagenesis to understand protein folding intermediates.
Related Concept Videos
01:22Protein Folding
Overview
01:25Protein Folding
Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation, critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.
Protein Structure Is Critical to Its Biological Function
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing...
Protein Structure Is Critical to Its Biological Function
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing...
03:00Molecular Chaperones and Protein Folding
The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...
The...
02:11Proteins: From Genes to Degradation
Within a biological system, the DNA encodes the RNA, and the nucleotide sequence in the RNA further defines the amino acid sequence in the protein. This is referred to as “The Central Dogma of Molecular Biology” - a term coined by Francis Crick. Central dogma is a firm principle in biology that defines the flow of genetic information within any life form. The two fundamental steps in central dogma are - transcription and translation.
Transcription is the synthesis of RNA molecules by RNA...
Transcription is the synthesis of RNA molecules by RNA...
01:57Gene Families
Gene families consist of groups of genes proposed to have originated from a common ancestor. Typically these arise through events in which a gene or genes are mistakenly duplicated during cell division. Unlike their parent genes (which are subject to selection pressure to maintain function), these gene copies do not need to preserve their sequences and may evolve at a relatively faster rate.
Occasionally these regions can be adapted to take on new roles within the organism, becoming novel genes...
Occasionally these regions can be adapted to take on new roles within the organism, becoming novel genes...
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...