Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1

Heidarsson, Pétur O.; Naqvi, Mohsin M.; Otazo, Mariela R.; Mossa, Alessandro; Kragelund, Birthe B.; Cecconi, Ciro

doi:10.1073/pnas.1401065111

Cited by 44 publications

(54 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These experiments revealed a sequential folding process where the C-domain first collapses into a partially folded structure and then undergoes a minor rearrangement to transit into its native conformation before the folding of the N-domain (11). We also showed that NCS-1 could become transiently kinetically trapped in two distinct misfolded states where pathologically high calcium concentrations increased the lifetimes of the misfolded structures (21). Although significant information is available on the structure, stability, and folding of NCS-1 in its Ca 2þ -bound state, little is still known about its structural and dynamical properties in the absence of Ca 2þ .…”

Section: Introductionmentioning

confidence: 76%

See 1 more Smart Citation

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

Naqvi

Heidarsson

Otazo

et al. 2015

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

Neuronal calcium sensor-1 (NCS-1) is the primordial member of a family of proteins responsible primarily for sensing changes in neuronal Ca(2+) concentration. NCS-1 is a multispecific protein interacting with a number of binding partners in both calcium-dependent and independent manners, and acting in a variety of cellular processes in which it has been linked to a number of disorders such as schizophrenia and autism. Despite extensive studies on the Ca(2+)-activated state of NCS proteins, little is known about the conformational dynamics of the Mg(2+)-bound and apo states, both of which are populated, at least transiently, at resting Ca(2+) conditions. Here, we used optical tweezers to study the folding behavior of individual NCS-1 molecules in the presence of Mg(2+) and in the absence of divalent ions. Under tension, the Mg(2+)-bound state of NCS-1 unfolds and refolds in a three-state process by populating one intermediate state consisting of a folded C-domain and an unfolded N-domain. The interconversion at equilibrium between the different molecular states populated by NCS-1 was monitored in real time through constant-force measurements and the energy landscapes underlying the observed transitions were reconstructed through hidden Markov model analysis. Unlike what has been observed with the Ca(2+)-bound state, the presence of Mg(2+) allows both the N- and C-domain to fold through all-or-none transitions with similar refolding rates. In the absence of divalent ions, NCS-1 unfolds and refolds reversibly in a two-state reaction involving only the C-domain, whereas the N-domain has no detectable transitions. Overall, the results allowed us to trace the progression of NCS-1 folding along its energy landscapes and provided a solid platform for understanding the conformational dynamics of similar EF-hand proteins.

show abstract

Section: Introductionmentioning

confidence: 76%

“…Likewise, the folding mechanism of NCS-1 in the presence of Ca 2þ has been studied extensively using both traditional ensemble methods (4,19) and single-molecule experiments with optical tweezers (11,21). Through mechanical manipulation, we have recently characterized the energy landscape of the Ca 2þ -bound state of NCS-1.…”

Section: Introductionmentioning

confidence: 99%

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

Naqvi

Heidarsson

Otazo

et al. 2015

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…A recent review provides details of studies that have employed the Zhurkov-Bell model to extract parameters of the unfolding energy landscape of a protein from experimental data on polyproteins [43]. In addition, since that review, 12 additional studies have used the Zhurkov-Bell model to extract information from force spectroscopy experiments [15,[44][45][46][47][48][49][50][51][52][53][54]. Given the prevalence of the Zhurkov-Bell model in force spectroscopy experiments, it is interesting to examine its application and accuracy in more detail.…”

Section: Introductionmentioning

confidence: 99%

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

et al. 2015

View full text Add to dashboard Cite

Single-molecule force spectroscopy using an atomic force microscope (AFM) can be used to measure the average unfolding force of proteins in a constant velocity experiment. In combination with Monte Carlo simulations and through the application of the Zhurkov-Bell model, information about the parameters describing the underlying unfolding energy landscape of the protein can be obtained. Using this approach, we have completed protein unfolding experiments on the polyprotein (I 27) 5 over a range of pulling velocities. In agreement with previous work, we find that the observed number of protein unfolding events observed in each approach-retract cycle varies between one and five, due to the nature of the interactions between the polyprotein, the AFM tip, and the substrate, and there is an unequal unfolding probability distribution. We have developed a Monte Carlo simulation that incorporates the impact of this unequal unfolding probability distribution on the median unfolding force and the calculation of the protein unfolding energy landscape parameters. These results show that while there is a significant, unequal unfolding probability distribution, the unfolding energy landscape parameters obtained from use of the Zhurkov-Bell model are not greatly affected. This result is important because it demonstrates that the minimum acceptance criteria typically used in force extension experiments are justified and do not skew the calculation of the unfolding energy landscape parameters. We further validate this approach by determining the error in the energy landscape parameters for two extreme cases, and we provide suggestions for methods that can be employed to increase the level of accuracy in single-molecule experiments using polyproteins.

show abstract

“…Although the in vitro folding of small and mediumsized proteins is relatively well understood (1)(2)(3)(4)(5), very limited information exists about the complete folding process of such large proteins (6). In general, larger proteins often exhibit a multitude of intermediate and aggregation-prone misfolded states (4,7). Recently, it has been shown that in multidomain proteins with homologous domains, cross-repeat intermediates can greatly slow down productive folding (8) but little is known about how size effects influence the folding of very large (>500 residues) nonhomologous multidomain proteins.…”

mentioning

confidence: 99%

Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments

Jahn

Büchner

Hugel

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of crossdomain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.misfolding | off-pathway | rough energy landscape | optical tweezers L arge protein machines consist of long amino acid chains, often exceeding many hundreds or even over a thousand residues in length. Although the in vitro folding of small and mediumsized proteins is relatively well understood (1-5), very limited information exists about the complete folding process of such large proteins (6). In general, larger proteins often exhibit a multitude of intermediate and aggregation-prone misfolded states (4, 7). Recently, it has been shown that in multidomain proteins with homologous domains, cross-repeat intermediates can greatly slow down productive folding (8) but little is known about how size effects influence the folding of very large (>500 residues) nonhomologous multidomain proteins.Methods providing dynamic as well as structural information are rare, and many bulk methods often do not provide enough resolution to deal with the multitude of states expected for complex systems such as the aforementioned large protein complexes. Single-molecule force spectroscopy offers kinetic, energetic as well as coarse primary structural information combined with the possibility of actively manipulating systems, making it ideally suited for studying the folding of large proteins (5,(9)(10)(11)(12).In this paper, we study the folding and assembly of the large chaperone machinery heat shock protein 90 from yeast (Hsp90), a protein that needs to fold and self-assemble before it can function as a chaperone in the cell. Hsp90 consists of three domains, the N-terminal domain (N domain, 211 residues), the middle domain (M domain, 266 residues), and the C-terminal domain (C domain, 172 residues). In eukaryotic Hsp90, the N and M domains are connecte...

show abstract

Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1

Cited by 44 publications

References 62 publications

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

Single-Molecule Folding Mechanisms of the apo- and Mg2+-Bound States of Human Neuronal Calcium Sensor-1

Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments

Contact Info

Product

Resources

About