Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome.

During the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of the Escherichia coli ribosomal protein S6 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

Evolutionary relationship of two ancient protein superfolds.

Proteins are the molecular machines of the cell that fold into specific three-dimensional structures to fulfill their functions. To improve our understanding of how the structure and function of proteins arises, it is crucial to understand how evolution has generated the structural diversity we observe today. Classically, proteins that adopt different folds are considered to be nonhomologous. However, using state-of-the-art tools for homology detection, we found evidence of homology between proteins of two ancient and highly populated protein folds, the (ßα)8-barrel and the flavodoxin-like fold. We detected a family of sequences that show intermediate features between both folds and determined what is to our knowledge the first representative crystal structure of one of its members, giving new insights into the evolutionary link of two of the earliest folds. Our findings contribute to an emergent vision where protein superfolds share common ancestry and encourage further approaches to complete the mapping of structure space onto sequence space.

Change in protein-ligand specificity through binding pocket grafting.

Recognition and discrimination of small molecules are crucial for biological processes in living systems. Understanding the mechanisms that underlie binding specificity is of particular interest to synthetic biology, e.g. the engineering of biosensors with de novo ligand affinities. Promising scaffolds for such biosensors are the periplasmic binding proteins (PBPs) due to their ligand-mediated structural change that can be translated into a physically measurable signal. In this study we focused on the two homologous polyamine binding proteins PotF and PotD. Despite their structural similarity, PotF and PotD have different binding specificities for the polyamines putrescine and spermidine. To elucidate how specificity is determined, we grafted the binding site of PotD onto PotF. The introduction of 7 mutations in the first shell of the binding pocket leads to a swap in the binding profile as confirmed by isothermal titration calorimetry. Furthermore, the 1.7Å crystal structure of the new variant complexed with spermidine reveals the interactions of the specificity determining residues including a defined water network. Altogether our study shows that specificity is encoded in the first shell residues of the PotF binding pocket and that transplantation of these residues allows the swap of the binding specificity.

Molecular handcraft of a well-folded protein chimera.

Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a repository of building blocks, known as domains, which trace back to even shorter segments that underwent numerous 'copy-paste' processes culminating in the scaffolds we see today. Utilizing the subdomain-database Fuzzle, we constructed a fold-chimera by integrating a flavodoxin-like fragment into a periplasmic binding protein. This chimera is well-folded and a crystal structure reveals stable interfaces between the fragments. These findings demonstrate the adaptability of α/ß-proteins and offer a stepping stone for optimization. By emphasizing the practicality of fragment databases, our work pioneers new pathways in protein engineering. Ultimately, the results substantiate the conjecture that periplasmic binding proteins originated from a flavodoxin-like ancestor.

A Short Tale of the Origin of Proteins and Ribosome Evolution.

Proteins are the workhorses of the cell and have been key players throughout the evolution of all organisms, from the origin of life to the present era. How might life have originated from the prebiotic chemistry of early Earth? This is one of the most intriguing unsolved questions in biology. Currently, however, it is generally accepted that amino acids, the building blocks of proteins, were abiotically available on primitive Earth, which would have made the formation of early peptides in a similar fashion possible. Peptides are likely to have coevolved with ancestral forms of RNA. The ribosome is the most evident product of this coevolution process, a sophisticated nanomachine that performs the synthesis of proteins codified in genomes. In this general review, we explore the evolution of proteins from their peptide origins to their folding and regulation based on the example of superoxide dismutase (SOD1), a key enzyme in oxygen metabolism on modern Earth.

Folding and Evolution of a Repeat Protein on the Ribosome.

Life on earth is the result of the work of proteins, the cellular nanomachines that fold into elaborated 3D structures to perform their functions. The ribosome synthesizes all the proteins of the biosphere, and many of them begin to fold during translation in a process known as cotranslational folding. In this work we discuss current advances of this field and provide computational and experimental data that highlight the role of ribosome in the evolution of protein structures. First, we used the sequence of the Ankyrin domain from the Drosophila Notch receptor to launch a deep sequence-based search. With this strategy, we found a conserved 33-residue motif shared by different protein folds. Then, to see how the vectorial addition of the motif would generate a full structure we measured the folding on the ribosome of the Ankyrin repeat protein. Not only the on-ribosome folding data is in full agreement with classical in vitro biophysical measurements but also it provides experimental evidence on how folded proteins could have evolved by duplication and fusion of smaller fragments in the RNA world. Overall, we discuss how the ribosomal exit tunnel could be conceptualized as an active site that is under evolutionary pressure to influence protein folding.

Identification and Analysis of Natural Building Blocks for Evolution-Guided Fragment-Based Protein Design.

Natural evolution has generated an impressively diverse protein universe via duplication and recombination from a set of protein fragments that served as building blocks. The application of these concepts to the design of new proteins using subdomain-sized fragments from different folds has proven to be experimentally successful. To better understand how evolution has shaped our protein universe, we performed an all-against-all comparison of protein domains representing all naturally existing folds and identified conserved homologous protein fragments. Overall, we found more than 1000 protein fragments of various lengths among different folds through similarity network analysis. These fragments are present in very different protein environments and represent versatile building blocks for protein design. These data are available in our web server called F(old P)uzzle (fuzzle.uni-bayreuth.de), which allows to individually filter the dataset and create customized networks for folds of interest. We believe that our results serve as an invaluable resource for structural and evolutionary biologists and as raw material for the design of custom-made proteins.

Cotranslational Folding of a Pentarepeat ß-Helix Protein.

It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a ß-helix pentarepeat protein from the human pathogen Clostridium botulinum-a homolog of the fluoroquinolone resistance protein MfpA-using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight ß-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is ~35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable ß-helix since it is structurally homologous to the smallest known ß-helix protein, a four-coil protein that is stable in solution.

Mutational analysis of protein folding inside the ribosome exit tunnel.

Recent work has demonstrated that cotranslational folding of proteins or protein domains in, or in the immediate vicinity of, the ribosome exit tunnel generates a pulling force on the nascent polypeptide chain that can be detected using a so-called translational arrest peptide (AP) engineered into the nascent chain as a force sensor. Here, we show that AP-based force measurements combined with systematic Ala and Trp scans of a zinc-finger domain that folds in the exit tunnel can be used to identify the residues that are critical for intraribosomal folding. Our results suggest a general approach to characterize the folded state(s) that may form as a protein domain moves progressively down the ribosome exit tunnel.