Details of the Partial Unfolding of T4 Lysozyme on Quartz Using Site‐Directed Spin Labeling

Jacobsen, Kerstin; Hubbell, Wayne L.; Ernst, Oliver P.; Risse, Thomas

doi:10.1002/anie.200600008

Cited by 30 publications

(12 citation statements)

References 44 publications

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“…Moreover, similar to the complexity in the mechanical unfolding pathways of T4L*, complex folding/unfolding intermediate states were also observed in chemical folding/unfolding of T4L* (40,(43)(44)(45)50 underlying energy landscape. It is likely that the unique two subdomain structure of T4L* and the associated domain-domain interactions play important roles in defining such a complex energy landscape that gives rise to the complex unfolding kinetics of T4L*.…”

Section: Discussionmentioning

confidence: 61%

“…Recent fragment studies confirmed the subdomain architecture of T4L* (18,45). In addition, a continuum of stability was observed by native-state hydrogen exchange to occur throughout each subdomain that may give rise to a variety of folding and unfolding intermediate states (40,43,44,50). In the mechanical unfolding of T4L*, we observed that the interaction between the N-terminal helix A and the reminder of the C-terminal lobe and the coupling between the two subdomains by helix A provide the dominant resistance to the mechanical unfolding.…”

Section: Discussionmentioning

confidence: 81%

See 1 more Smart Citation

Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: Evidence for a kinetic partitioning mechanism

Peng

2008

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

109

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Kinetic partitioning is predicted to be a general mechanism for proteins to fold into their well defined native three-dimensional structure from unfolded states following multiple folding pathways. However, experimental evidence supporting this mechanism is still limited. By using single-molecule atomic force microscopy, here we report experimental evidence supporting the kinetic partitioning mechanism for mechanical unfolding of T4 lysozyme, a small protein composed of two subdomains. We observed that on stretching from its N and C termini, T4 lysozyme unfolds by multiple distinct unfolding pathways: the majority of T4 lysozymes unfold in an all-or-none fashion by overcoming a dominant unfolding kinetic barrier; and a small fraction of T4 lysozymes unfold in three-state fashion involving unfolding intermediate states. The three-state unfolding pathways do not follow well defined routes, instead they display variability and diversity in individual unfolding pathways. The unfolding intermediate states are local energy minima along the mechanical unfolding pathways and are likely to result from the residual structures present in the two subdomains after crossing the main unfolding barrier. These results provide direct evidence for the kinetic partitioning of the mechanical unfolding pathways of T4 lysozyme, and the complex unfolding behaviors reflect the stochastic nature of kinetic barrier rupture in mechanical unfolding processes. Our results demonstrate that single-molecule atomic force microscopy is an ideal tool to investigate the folding/unfolding dynamics of complex multimodule proteins that are otherwise difficult to study using traditional methods.energy landscape ͉ force spectroscopy ͉ multiple pathways ͉ single-molecule studies ͉ unfolding intermediate P rotein folding and unfolding are fundamental processes inside the cell. From an unfolded and presumably random coil-like state, most proteins must fold into the well defined threedimensional structures, which are unique to each protein, to be biologically functional. The folding and unfolding processes are complex and may involve multiple pathways (1, 2), which are believed to be governed by the general kinetic partitioning mechanism (3, 4), although experimental proof supporting this mechanism is still limited (5-8). Small, single-domain proteins are often used as model systems to investigate folding/unfolding dynamics. However, certain proteins are complex such that they are composed of smaller modules that can behave quite independently, thus presenting more complex folding/unfolding dynamics. The coupling between modules plays important roles in defining the overall conformational dynamics of these proteins (9-11). T4 lysozyme is an excellent model system in this aspect. T4 lysozyme is 164 residues long and composed of 10 ␣-helices and four short ␤-strands (12) (Fig. 1A). It has been widely studied for Ͼ30 years and the availability of high-resolution structures of hundreds of T4 lysozyme mutants makes it especially appealing (13). Although traditio...

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Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 81%

Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: Evidence for a kinetic partitioning mechanism

Peng

2008

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

109

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“…Previously T4L, when adsorbed at solid surface such as quartz 44 or silica nanoparticles 45 , was found to become partially unfolded and lose its enzymatic activity, but the present study indicates that T4L retains its conformation when adsorbed inside nanochannels. Therefore, it is important to perform a careful inspection of the protein properties, when proteins are loaded onto surface of nanoparticles or into nanochannels, using the demonstration methods of the present study.…”

Section: Discussionmentioning

confidence: 47%

Study of Protein Dynamics under Nanoconfinement by Spin-Label ESR: A Case of T4 Lysozyme Protein

2017

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The electron spin resonance (ESR) spectra of spin-labeled proteins are sensitive to dynamics, but discrimination between the various dynamics is often difficult. Here, we report an improvement in ESR spectral sensitivity to local backbone dynamics of a protein by a methodology that performs ESR measurement when the protein is confined in the nanochannels of a mesoporous material. An extensive set of ESR data, which includes the spectra of a nitroxide-based side chain from buried and solvent-exposed sites of a T4 lysozyme (T4L) protein, were obtained over a range of temperatures, 200-300 K, to explore the dynamics of T4L under nanoconfinement. Spectra were simulated by performing theoretical fits to the data using the microscopic ordering with macroscopic disordering model. Two principle dynamic modes, which differ in mobility and ordering, are required to account for the spectra at temperatures >240 K. We show that the mobile one correlates only with the local backbone dynamics of buried sites, whereas the other reflects the difference in local hydration dynamics between the labeling sites in T4L. The assignment of the mobile component is supported by the X-ray crystallography data of T4L. Collectively, this study has demonstrated the validity of such a methodology for improving ESR sensitivity to buried sites in a protein.

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“…Only in the past decade it was shown that it can be extended to study proteins interaction with solid surfaces [26,27]. Recently published studies demonstrated that this method can be applicable to proteins encapsulated [15] in or adsorbed on solid surfaces [28][29][30][31][32], thus permitting investigation of adsorption-induced conformational changes in conditions close to the proteins physiological environment (phosphate buffer solution at pH 7.4).…”

Section: Methods Used For Studying Proteins Interaction With Solid Sumentioning

confidence: 98%

Glass-Ceramics: Fundamental Aspects Regarding the Interaction with Proteins

Gruian¹,

Vanea²,

Steinhoff³

et al. 2015

Handbook of Bioceramics and Biocomposites

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Details of the Partial Unfolding of T4 Lysozyme on Quartz Using Site‐Directed Spin Labeling

Cited by 30 publications

References 44 publications

Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: Evidence for a kinetic partitioning mechanism

Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: Evidence for a kinetic partitioning mechanism

Study of Protein Dynamics under Nanoconfinement by Spin-Label ESR: A Case of T4 Lysozyme Protein

Glass-Ceramics: Fundamental Aspects Regarding the Interaction with Proteins

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