Albert Galera-Prat

Understanding biology by stretching proteins: recent progress

Single molecule manipulation techniques combined with molecular dynamics simulations and protein engineering have enabled, during the last decade, the mechanical properties of proteins to be studied directly, thereby giving birth to the field of protein nanomechanics. Recent data obtained from such techniques have helped gain insight into the structural bases of protein resistance against forced unfolding, as well as revealing structural motifs involved in mechanical stability. Also, important technical developments have provided new perspectives into protein folding. Eventually, new and exciting data have shown that mechanical properties are key factors in cell signaling and pathologies, and have been used to rationally tune these properties in a variety of proteins.

Common Features at the Start of the Neurodegeneration Cascade

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis.

Molecular Basis of Orb2 Amyloidogenesis and Blockade of Memory Consolidation

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.

Theoretical tests of the mechanical protection strategy in protein nanomechanics

We provide theoretical tests of a novel experimental technique to determine mechanostability of proteins based on stretching a mechanically protected protein by single‐molecule force spectroscopy. This technique involves stretching a homogeneous or heterogeneous chain of reference proteins (single‐molecule markers) in which one of them acts as host to the guest protein under study. The guest protein is grafted into the host through genetic engineering. It is expected that unraveling of the host precedes the unraveling of the guest removing ambiguities in the reading of the force‐extension patterns of the guest protein. We study examples of such systems within a coarse‐grained structure‐based model. We consider systems with various ratios of mechanostability for the host and guest molecules and compare them to experimental results involving cohesin I as the guest molecule. For a comparison, we also study the force‐displacement patterns in proteins that are linked in a serial fashion. We find that the mechanostability of the guest is similar to that of the isolated or serially linked protein. We also demonstrate that the ideal configuration of this strategy would be one in which the host is much more mechanostable than the single‐molecule markers. We finally show that it is troublesome to use the highly stable cystine knot proteins as a host to graft a guest in stretching studies because this would involve a cleaving procedure. Proteins 2014; 82:717–726.

The Y9P variant of the titin I27 module: Structural determinants of its revisited nanomechanics.

The titin I27 module from human cardiac titin has become a standard in protein nanomechanics. A proline-scanning study of its mechanical clamp found three mechanically hypomorphic mutants and a paradoxically hypermorphic mutant (I27Y9P). Both types of mutants have been commonly used as substrates of several protein unfoldase machineries in studies relating protein mechanostability to translocation or degradation rates. Using single-molecule force spectroscopy based on atomic force microscopy, polyprotein engineering, and steered molecular dynamics simulations, we show that, unexpectedly, the mechanostability of the Y9P variant is comparable to the wild type. Furthermore, the NMR analysis of homomeric polyproteins of this variant suggests that these constructs may induce slight structural perturbations in the monomer, which may explain some minor differences in this variants properties; namely the abolishment of the mechanical unfolding intermediate and a reduced thermal stability. Our results clarify a previously reported paradoxical result in protein nanomechanics and contribute to refining our toolbox for understanding the unfolding mechanism used by translocases and degradation machines.

The cohesin module is a major determinant of cellulosome mechanical stability

Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.

Non-local effects of point mutations on the stability of a protein module

We combine experimental and theoretical methods to assess the effect of a set of point mutations on c7A, a highly mechanostable type I cohesin module from scaffoldin CipA from Clostridium thermocellum. We propose a novel robust and computationally expedient theoretical method to determine the effects of point mutations on protein structure and stability. We use all-atom simulations to predict structural shifts with respect to the native protein and then analyze the mutants using a coarse-grained model. We examine transitions in contacts between residues and find that changes in the contact map usually involve a non-local component that can extend up to 50 Å. We have identified mutations that may lead to a substantial increase in mechanical and thermodynamic stabilities by making systematic substitutions into alanine and phenylalanine in c7A. Experimental measurements of the mechanical stability and circular dichroism data agree qualitatively with the predictions provided the thermal stability is calculated using only the contacts within the secondary structures.

Transcytosis in the blood–cerebrospinal fluid barrier of the mouse brain with an engineered receptor/ligand system

Crossing the blood–brain and the blood–cerebrospinal fluid barriers (BCSFB) is one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because these barriers prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Based on this mechanism, we engineered a receptor/ligand system to overcome the brain barriers by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested the hybrid receptor in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the BCSFB, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.

Solution conformation of a cohesin module and its scaffoldin linker from a prototypical cellulosome

Bacterial cellulases are drawing increased attention as a means to obtain plentiful chemical feedstocks and fuels from renewable lignocellulosic biomass sources. Certain bacteria deploy a large extracellular multi-protein complex, called the cellulosome, to degrade cellulose. Scaffoldin, a key non-catalytic cellulosome component, is a large protein containing a cellulose-specific carbohydrate-binding module and several cohesin modules which bind and organize the hydrolytic enzymes. Despite the importance of the structure and protein/protein interactions of the cohesin module in the cellulosome, its structure in solution has remained unknown to date. Here, we report the backbone 1H, 13C and 15N NMR assignments of the Cohesin module 5 from the highly stable and active cellulosome from Clostridium thermocellum. These data reveal that this module adopts a tightly packed, well folded and rigid structure in solution. Furthermore, since in scaffoldin, the cohesin modules are connected by linkers we have also characterized the conformation of a representative linker segment using NMR spectroscopy. Analysis of its chemical shift values revealed that this linker is rather stiff and tends to adopt extended conformations. This suggests that the scaffoldin linkers act to minimize interactions between cohesin modules. These results pave the way towards solution studies on cohesin/dockerins fascinating dual-binding mode.

Nanomechanics of Proteins, Both Folded and Disordered

Single-molecule techniques have recently provided a versatile tool for imaging and manipulating protein molecules one at a time, enabling us to address important biological questions in key areas of cell biology (e.g., cell adhesion and signaling, neurodegeneration) and protein science (e.g., protein folding, protein structure and stability, catalysis, protein evolution, conformational polymorphism, and amyloidogenesis). One of these techniques, single-molecule force spectroscopy based on atomic force microscopy, combined with theoretical/computational approaches and protein engineering, has allowed unprecedented progress in characterizing and understanding at the molecular level the mechanical properties of biomolecules, particularly those of proteins, which has recently opened the new, exciting and fast-growing research field of protein nanomechanics. The aim of this review is to describe the principles of this methodology and to discuss the main achievements in this field, with special emphasis on its emerging application to the analysis of intrinsically disordered proteins.