The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations

Caldarini, Martina; Sonar, Punam; Valpapuram, Immanuel; Tavella, Davide; Volonté, Cinza; Pandini, V.; Vanoni, Maria A.; Aliverti, Alessandro; Broglia, R. A.; Tiana, Guido; Cecconi, Ciro

doi:10.1016/j.bpc.2014.08.001

Cited by 19 publications

(16 citation statements)

References 51 publications

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“…on proteins. Although the small size of usual proteins may pose experimental challenge, large filamentous proteins (e.g., titin [50]) or proteins captured with DNA handles [51][52][53][54] may be investigated directly. Considering the emerging significance of single-molecule mechanics in understanding structural and functional detail, the addition of precisely adjusted ligand concentration gradients, as demonstrated here, may provide further access to understanding the exact mechanisms behind biomolecular phenomena.…”

Section: Discussionmentioning

confidence: 99%

Single-Molecule Mechanics in Ligand Concentration Gradient

et al. 2020

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Single-molecule experiments provide unique insights into the mechanisms of biomolecular phenomena. However, because varying the concentration of a solute usually requires the exchange of the entire solution around the molecule, ligand-concentration-dependent measurements on the same molecule pose a challenge. In the present work we exploited the fact that a diffusion-dependent concentration gradient arises in a laminar-flow microfluidic device, which may be utilized for controlling the concentration of the ligand that the mechanically manipulated single molecule is exposed to. We tested this experimental approach by exposing a λ-phage dsDNA molecule, held with a double-trap optical tweezers instrument, to diffusionally-controlled concentrations of SYTOX Orange (SxO) and tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP). We demonstrate that the experimental design allows access to transient-kinetic, equilibrium and ligand-concentration-dependent mechanical experiments on the very same single molecule.

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Section: Discussionmentioning

confidence: 99%

Single-Molecule Mechanics in Ligand Concentration Gradient

et al. 2020

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“…Marqusee and colleagues probed the properties of different stages of folding, showing that molten globules can be distinguished from fully native states through their increased compliance [30] and extending classic phi analysis of transition states into the single-molecule regime [31]. Measurements of the unfolding and refolding of monomeric HIV-1-protease validated simulations suggesting the existence of multiple pathways [32], revealing not only two-state unfolding but also unfolding through an intermediate and an ensemble of partially folded states en route to the native state, which themselves unfolded 44 Biophysical and molecular biological methods via multiple pathways [33]. Studies of SNARE complex assembly, which involves a stable four-helix bundle, helped clarify how it drives membrane fusion to allow transport of molecules between different membranes [34,35] and identified rare misfolded states of coiled-coils [36] that may be involved in diseases related to SNARE misfolding [37].…”

Section: Introductionmentioning

confidence: 97%

Probing the structural dynamics of proteins and nucleic acids with optical tweezers

Ritchie

Woodside

2015

Current Opinion in Structural Biology

112

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Conformational changes are an essential feature of most molecular processes in biology. Optical tweezers have emerged as a powerful tool for probing conformational dynamics at the single-molecule level because of their high resolution and sensitivity, opening new windows on phenomena ranging from folding and ligand binding to enzyme function, molecular machines, and protein aggregation. By measuring conformational changes induced in a molecule by forces applied by optical tweezers, new insight has been gained into the relationship between dynamics and function. We discuss recent advances from studies of how structure forms in proteins and RNA, including non-native structures, fluctuations in disordered proteins, and interactions with chaperones assisting native folding. We also review the development of assays probing the dynamics of complex protein-nucleic acid and protein-protein assemblies that reveal the dynamic interactions between biomolecular machines and their substrates.

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“…Under tension, the unfolding and refolding of a protein is accompanied by changes in the extension of the molecule, giving rise to discontinuities (rips) in the recorded force traces. A molecule can be stretched and relaxed at constant speed (Heidarsson et al, 2012b; Caldarini et al, 2014), or it can be kept at a specific force through a force feedback mechanism and observed to fluctuate between different molecular conformations (Heidarsson et al, 2013b). A careful analysis of the experimental data and the use of advanced statistical methods allow a detailed characterization of the folding and, possibly, misfolding processes of the molecule and the reconstruction of its energy landscape (Stigler et al, 2011).…”

Section: A Single Molecule Perspective On Protein Folding Using Direcmentioning

confidence: 99%

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

Choudhary

Kragelund

Heidarsson

et al. 2018

Front. Mol. Neurosci.

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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.

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The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations

Cited by 19 publications

References 51 publications

Single-Molecule Mechanics in Ligand Concentration Gradient

Single-Molecule Mechanics in Ligand Concentration Gradient

Probing the structural dynamics of proteins and nucleic acids with optical tweezers

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

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