Dissertation
Designing and understanding metal-selective proteins
Transition metals play an essential role in biological systems, where approximately one-third of all proteins depend on a metal cofactor to perform their function.
- Author
- B. Rooijakkers
- Date
- 19 May 2026
- Links
- Thesis in Leiden Repository
In cells, incorporation of the correct metal ion into a specific protein is governed by multiple factors, like protein structure, the Irving-Williams series, strict control over the local metal concentration, compartmentalization and presence of metallochaperones. Often, proteins exhibit metal promiscuity when taken out of their cellular environment, meaning that they are able to bind multiple types of metals other than their cognate metal ion. Metal selectivity is therefore an interesting property to study. This can be done through de novo protein design, which focuses on the design of proteins from scratch or from first principles. De novo protein design makes use of simplified protein structures, which makes it easier to elucidate structure-function relationships. This approach allows construction of proteins with a defined structure and minimal sequence complexity, providing a simplified framework to study the intrinsic contributions of structure to metal coordination, independent of cellular metal regulation.
This work investigates how de novo designed coiled coil protein scaffolds can be engineered to fold and bind specific transition metal ions, and how their architecture influences metal dependent stability and metal selectivity. In chapter 2 a single-chain, three-helix protein termed 3hC was designed and characterized to examine how structural features determine metal binding. Biophysical analyses revealed that 3hC is unfolded in the absence of metal ions but folds upon addition of Co(II), Ni(II), Zn(II), or Cu(II). Thermal unfolding experiments showed that the Ni(II)-bound complex was the most stable, whereas Cu(II) formed the less stable complex, which goes against the general trend of the Irving-Williams series. Native mass spectrometry further indicated metal-dependent differences in stoichiometry and coordination, suggesting that the compatibility between the metal type, the coordination geometry and the protein fold dictates overall stability. In chapter 3, to explore the effect of scaffold topology on metal binding, a protein variant named 3hN was designed by relocating the metal-binding site in 3hC from the C- to the N-terminus of the protein. This modification altered both metal preferences and thermal stability, demonstrating that structural rearrangements of the protein can shift metal-binding behavior. Introduction of an N-capping box in the interhelical loops enhanced metal-dependent thermal stability, but solely for Co(II)- and Ni(II)-complexes, and solely when the metal-binding site was located at the N-terminus, underscoring the sensitivity of metal coordination to local secondary structure context. Finally, in chapter 4, a proof-of-concept application was developed by coupling metal-induced folding to an enzymatic output using a β-lactamase–β-lactamase inhibitory protein (BLA-BLIP) sensor system. Constructs that incorporated 3hN exhibited a two-fold activation response with Cu(II), demonstrating that metal-dependent folding can be functionally translated into a measurable enzymatic signal. This system can be used in cells for a directed evolution approach where we screen for mutants that comprise of high metal binding affinity and metal selectivity.
Together, these findings establish structure-function relationships governing metal binding in de novo designed proteins and provide the first step of a proof-of-concept for combing de novo protein design with directed evolution in order to screen for metal-selective proteins.