Selective Excitation of Native Fluctuations during Thermal Unfolding Simulations: Horse Heart Cytochrome c as a Case Study

Roccatano, Danilo; Daidone, Isabella; Ceruso, Marc-Antoine; Bossa, Cecilia; Nola, Alfredo Di

doi:10.1016/s0006-3495(03)74995-9

Cited by 46 publications

(28 citation statements)

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“…Once the N-C foldon is locked in place, it guides the folding of the rest of the protein. Simulations also support this sequential stepwise folding pathway (17,18). The N-C foldon is crucial for the stability of the whole protein and mutations in either the N-or C-terminal helix significantly affects folding (26,27).…”

Section: Np Destabilizes the Whole Protein When Attached On Structuresmentioning

confidence: 81%

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Site-directed nanoparticle labeling of cytochrome c

Aubin‐Tam

Hwang

Hamad‐Schifferli

2009

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

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Although nanoparticle-protein conjugates have been synthesized for numerous applications, bioconjugation remains a challenge, often resulting in denaturation or loss of protein function. This is partly because the protein-nanoparticle interface is poorly understood, which impedes the use of nanoparticles in nanomedicine. Although the effects of nanoparticle ligand and material on protein structure have been explored, the choice of the labeling site on the protein has not yet been systematically studied. To address this issue, we label cytochrome c site-specifically with a negatively charged Au nanoparticle via a covalent thiol-Au bond. The attachment site is controlled by cysteine mutations of surface residues. The effect of labeling on protein structure is probed by circular dichroism. Protein unfolding is the most severe when the nanoparticle is attached to the N-and C-terminal foldon, the core motif of cytochrome c. Also, when the nanoparticle is attached in the vicinity of charged residues, the amount of structural damage is greater because of salt-dependent electrostatic interactions with charged ligand bis(p-sulfonatophenyl) phenylphosphine on the nanoparticle. Molecular dynamics simulations also elucidate local to global structural perturbation depending on labeling site. These results suggest that the labeling site must be considered as one of the main design criteria for nanoparticle-protein conjugates.bioconjugation ͉ protein folding ͉ circular dichroism ͉ molecular dynamics simulation ͉ nanoscale interface N anomaterials hold much promise for biomedical applications such as multiscale imaging (1-3), novel therapeutic approaches (4), and biomolecular sensing (5). The emerging field of nanomedicine is based on exploiting the unique size-and material-dependent properties of nanomaterials. However, the biggest barrier in effectively using them is their biological interface. Historically, biological-inorganic interfaces suffer complications such as surface fouling in medical devices and nonspecific adsorption of proteins on sensors, inhibiting practical use. The problem significantly worsens for nanoscale systems because of their high surface-to-volume ratio. Nanoscale surfaces are physically and chemically different from the bulk, requiring nontraditional tools to effectively probe them. These factors suggest a pressing need for deeper understanding of the ''nano-bio'' interface.One particular issue related to the interface that limits the potential of nanoparticles (NPs) is conjugation to a biomolecule. Covalently linking a NP to a protein can affect both its structure and function. Even if protein structure is retained, the NP can sterically hinder substrate binding. NPs are large compared with dye molecules, and their interface with proteins is highly complex. NPs are not hard spheres but crystals with facets, edges, and vertices, and their passivating ligands can change conformation or come on and off the NP. Consequently, there are numerous nonspecific interactions between the NP and protein, where a...

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Section: Np Destabilizes the Whole Protein When Attached On Structuresmentioning

confidence: 81%

“…The heme was covalently linked to Cys 14 and 17. Covalent bonds were maintained between the heme iron and Met-80 and His-18, which are assumed to remain formed during the timescale of our simulations (17). Cyt c was linked to the NP surface by replacing one BPS ligand .…”

Section: Conjugation Of Cyt C With Npsmentioning

confidence: 99%

Site-directed nanoparticle labeling of cytochrome c

Aubin‐Tam

Hwang

Hamad‐Schifferli

2009

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

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“…Essential dynamics has been used to look at the native-state fluctuations of proteins (Ceruso et al 1999;Merlino et al 2003;Merlino et al 2004) as well as thermal denaturation trajectories (Roccatano et al 2003). It has also proven useful in the identification of protein folding transition state ensembles (Marianayagam & Jackson 2004).…”

Section: Introductionmentioning

confidence: 99%

Native-state dynamics of the ubiquitin family: implications for function and evolution

Marianayagam

Jackson

2005

J. R. Soc. Interface.

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Protein dynamics are integral to protein function. In recent years, the use of computer simulation to understand the molecular motions of proteins has become widespread. However, there are few such studies which compare the dynamics of proteins that are structurally and functionally related. In this study, we present native-state molecular dynamic simulations of four proteins which possess a ubiquitin-like fold. Three of these proteins are thought to have evolved from a common ancestral ubiquitin-like protein and have similarities in their function. A fourth protein, which is structurally homologous but which appears to have a different function, is also studied. Local fluctuations in the native state simulations are analysed, and conserved motions of the C-a backbone atoms are identified in residues which are important for function. In addition, the global dynamics of the proteins are analysed using the essential-dynamics method. This analysis reveals a slightly higher degree of conservation in dynamics for the three proteins which are functionally related. Both the global and local analyses illustrate how nature has optimized and conserved protein motions for specific biological activity within the ubiquitin family.

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“…These data revealed that the stability of holo cyt c's increased upon mutation. From the crystal structure, the R g (protein) value for holo WT cyt c is 1.26 nm [28].…”

Section: Simulations Of Y67f and F82h With Feá á áS (Met-80) Bondmentioning

confidence: 99%