The Complex Conformational Dynamics of Neuronal Calcium Sensor-1: A Single Molecule Perspective

The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca2+ changes in neurons and is linked to a wide range of neuronal functions such as regulation of neurotransmitter release, voltage-gated Ca2+ channels and neuronal outgrowth. Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca2+-bound, Mg2+-bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca2+ and Mg2+) to its EF-hands. At pathologically high Ca2+ concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties

Bio-molecular applications of recent developments in optical tweezers

In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT\u2019s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT

The complex conformational dynamics of neuronal calcium sensor-1: A single molecule perspective

The human neuronal calcium sensor-1 (NCS-1) is a multispecific two-domain EF-hand protein expressed predominantly in neurons and is a member of the NCS protein family. Structure-function relationships of NCS-1 have been extensively studied showing that conformational dynamics linked to diverse ion-binding is important to its function. NCS-1 transduces Ca 2+ changes in neurons and is linked to a wide range of neuronal functions such as regulation of neurotransmitter release, voltage-gated Ca 2+ channels and neuronal outgrowth. Defective NCS-1 can be deleterious to cells and has been linked to serious neuronal disorders like autism. Here, we review recent studies describing at the single molecule level the structural and mechanistic details of the folding and misfolding processes of the non-myristoylated NCS-1. By manipulating one molecule at a time with optical tweezers, the conformational equilibria of the Ca 2+ -bound, Mg 2+ -bound and apo states of NCS-1 were investigated revealing a complex folding mechanism underlain by a rugged and multidimensional energy landscape. The molecular rearrangements that NCS-1 undergoes to transit from one conformation to another and the energetics of these reactions are tightly regulated by the binding of divalent ions (Ca 2+ and Mg 2+ ) to its EF-hands. At pathologically high Ca 2+ concentrations the protein sometimes follows non-productive misfolding pathways leading to kinetically trapped and potentially harmful misfolded conformations. We discuss the significance of these misfolding events as well as the role of inter-domain interactions in shaping the energy landscape and ultimately the biological function of NCS-1. The conformational equilibria of NCS-1 are also compared to those of calmodulin (CaM) and differences and similarities in the behavior of these proteins are rationalized in terms of structural properties

Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process

: Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity

Studio a livello di singola molecola del folding, misfolding e aggregazione di proteine e dell’attività chaperonica della HSPB8

Negli ultimi decenni le pinze ottiche si sono rivelate una tecnica sperimentale estremamente efficace per eseguire studi di spettroscopia di forza a livello di singola molecola. In particolare, un’applicazione delle pinze ottiche che sta avendo una rilevanza biomedica sempre più importante è quella relativa allo studio dei processi di ripiegamento corretto (folding), non corretto (misfolding) e dell’aggregazione di proteine. Di forte rilevanza biomedica è anche la possibilità offerta dalle pinze ottiche di caratterizzare in grande dettaglio i meccanismi molecolari che mediano le interazioni tra due o più biomolecole, come ad esempio tra uno chaperone molecolare e il suo substrato. La rilevanza medica di questi studi deriva dal fatto che l'errato ripiegamento e l'aggregazione delle proteine sono processi deleteri, spesso associati a neurodegenerazione. Gli chaperoni molecolari si sono evoluti come strumento molecolare per combattere sia il misfolding che l’aggregazione proteica. Un funzionamento non corretto degli chaperoni molecolari spesso causa perdita di proteostasi e l’insorgenza di varie patologie umane. Il lavoro descritto in questa tesi spiega in maniera dettagliata l’approccio sperimentale utilizzato per utilizzare le pinze ottiche per lo studio del folding, misfolding e aggregazione di proteine. In particolare in questa tesi vengono descritti: i) i risultati di esperimenti mirati alla elucidazione del processo di ripiegamento corretto e non del sensore al calcio NCS-1 (Neuronal Calcium Sensor 1; ii) l'approccio sperimentale adottato per descrivere la dinamica strutturale e funzionale di vari chaperoni molecolari utilizzando le pinze ottiche e la microscopia a forza atomica; iii) recenti sviluppi tecnici che hanno ampliato le possibili applicazioni delle pinze ottiche in campo biologico; iv) i risultati di esperimenti mirati a far luce sui meccanismi molecolari che mediano l’attività chaperonica dello chaperone molecolare HSPB8. In quest’ultimi esperimenti abbiamo manipolato meccanicamente monomeri e tetrameri della Maltose Binding Protein (MBP) e analizzato i loro processi di folding, misfolding e aggregazione in presenza e assenza del HSPB8 wild-type e del suo mutante HSPB8-K141E. I nostri risultati dimostrano una forte attività antiaggregante (holdase activity) della HSPB8 che riduce significativamente l'aggregazione delle molecole di MBP e un’attività antiaggregante molto ridotta del mutante HSPB8-K141E. Inoltre, i nostri studi rivelano una inaspettata attività pro-folding (foldase activity) sia della forma mutata che di quella wild-type della HSPB8. Questi dati sperimentali evidenziano nuovi meccanismi di interazione tra HSPB8 e i suoi substrati e suggeriscono un ruolo fisiologico più complesso per questo chaperone molecolare di quanto precedentemente ipotizzato.Optical tweezers have evolved as an exemplary Single Molecule Force Spectroscopy (SMFS) technique over the past three decades. A distinct and bio medically relevant application of Optical Tweezers is their ability to observe directly at single molecule level the folding, misfolding and aggregation of protein molecules. Additionally the dynamic approach of Optical Tweezer setup also allows for the isolated study of interactions between two or more biomolecules, such as chaperone-protein interactions, in real time. The medical relevance of such studies stems from the fact that misfolding and aggregation of proteins are deleterious processes and have been linked to many neurodegenerative disorders. While molecular chaperones have evolved as an evolutionarily conserved sword and shield mechanism against such deleterious processes, wherein their holdase action acts as a shield preventing further aggregation of misfolded protein species and their foldase action acts as a sword and actively assists misfolded structure to regains their natively folded state. The dysfunction of this chaperone activity is also cytotoxic and can lead to loss of proteostasis. The present thesis dwells deeper in this specific application of Optical tweezer. The thesis will elaborate upon how optical tweezers can extract the mechanistic details of the folding and misfolding of protein molecules by reviewing the experiments performed on NCS-1 (Neuronal Calcium Sensor 1). It will also discuss the experimental approach taken by SMFS techniques like Optical Tweezers and AFM (Atomic Force Microscopy) to study the structural and functional dynamics of molecular chaperones. Furthermore, the thesis will explore the recent developments in Optical Tweezers and their biological applications. Finally, I describe the results of experiments we have carried out on the maltose binding protein to elucidate the mechanism of action of the chaperone HSPB8. We have mechanically denatured homotetramers of MBP as well as single MBP molecules and analyzed their folding and aggregation processes in the presence and absence of wild-type HSPB8 and its mutant form HSPB8-K141E/N. Our results reveal a strong holdase activity of wild type HSPB8, which either prevents completely the aggregation of denatured MBP molecules or allows the substrate to form only small and mechanically weak aggregates while this holdase activity is significantly suppressed in the mutant. Moreover, and importantly, a careful analysis of the data also discloses an unexpected foldase activity of both wild type and mutated forms of HSPB8, which guides the folding process of denatured MBP molecules into their native states. Our findings highlight new mechanisms of interaction between HSPB8 and its substrates and suggest a more complex physiological role for this chaperone than previously assumed