The usual term is "structure-function relationships", and instead of structures at the atomic level we probe conformation (all changes) and stability of proteins in intact cells. This research line forms the basis for the other lines. Because of our in-depth knowledge of the folding of our model proteins we exploit them to study cellular processes and responses. We have characterized the basic folding pathways for several proteins in the endoplasmic reticulum (ER), the first compartment of the secretory pathway: the glycoproteins of influenza virus and HIV-1, the LDL receptor (a repeat protein), and the ABC-transporter CFTR, mutated in cystic fibrosis patients. Disulfide bonds, resistance to proteolytic digestion, antigenic epitopes, and glycosylation are examples of the features we use to probe folding. We for instance found that incorrect, inter-repeat disulfide bonds are obligatory intermediates during efficient folding of the LDL receptor rather than a road to misfolding, and that calcium incorporation is essential for the native phase of folding. We have several manuscripts in preparation that report on the interplay between the repeats within the LDL receptor and the role of this folding path for LDL receptor function, which suggests similar complex but essential folding pathways for other repeat protein families. Aim is to establish how the complete ER environment (from calcium, ATP, redox to folding factors) manages this folding pathway, preventing initial native folding of the repeats and supporting it at a later stage.
An exciting project in progress is the folding of CFTR and the defect in cystic fibrosis mutants. We showed before that the individual membrane-spanning (MSDs) and nucleotide-binding domains (NBDs) fold co-translationally, followed by a largely post-translational domain assembly phase. Rather than in the expected (as it contains the major patient mutation F508del) subdomain in NBD1, the defect appears in the integration of discontinuous up- and downstream subdomains, a folding pathway reminiscent of our yet unpublished influenza virus hemagglutinin folding path. This and other similarities suggest a general principle of proteins folding as hairpins, zippering up from core to periphery, irrespective of protein sequence and structure, a suggestion we will follow up with experiment. Understanding the folding path now allows an understanding of the mechanism of action of corrector drugs and molecular chaperones on the folding protein (see research line below). Our future focus is the role of intramembrane, ER-luminal, and cytosolic chaperoning during (multimembrane-spanning) domain assembly and the importance of the membrane (thickness) for proper assembly in the ER. For both LDL receptor and CFTR we have started examining the role of local translation rate on protein folding, by changing silent codons.
To relate folding to function we have used reverse genetics for HIV-1 (inserting mutant Env into the virus) in combination with in-vitro evolution, a powerful approach that teaches us how the virus solves a folding problem and escapes the defect. This marks events on the folding pathway, and also identifies the insults the virus can nót escape from. We intend to use the same approach for influenza virus. Next to common features, each protein shows peculiarities, such as the late, folding-dependent cleavage of the signal peptide that targets HIV Env to the secretory pathway. We are preparing 2 manuscripts that will describe the mechanism and function of this conserved process. It is a fascinating story of timing and regulation through conformational changes. Whereas signal peptide cleavage of Env bears all hallmarks of Signal Peptidase, we will examine its role as well as that of Signal Peptide Peptidase and other candidate intramembrane chaperones (see below).
One of the novel folding assays we established bridges the gap between in vitro refolding and folding in intact cells, with the aim to reconstitute the folding process in vitro. We have now managed to successfully fold influenza virus hemagglutinin in a diluted detergent cell lysate through addition of the proper redox buffer and excess protein disulfide isomerase, and plan to use this for further reconstitution and characterization of the folding process at the level of the folding molecule.
This research line offers detailed insights into folding of proteins in intact cells, its requirements, and its relation to protein function. It generates new (folding) assays, tools, and reagents and so far has allowed us to determine the molecular target of a corrector drug in the clinical pipeline for cystic fibrosis, design of assays suitable for high-throughput screening for antiviral or CFTR corrector compounds, and the partial reconstitution of folding of a complex protein without an intact ER.