Biofunctionalized, Ultrathin Coatings of Cross-Linked Star-Shaped Poly(ethylene oxide) Allow Reversible Folding of Immobilized Proteins

Groll, Jürgen; and, Elza V. Amirgoulova,#,†; Ameringer, Thomas; Heyes, Colin D.; Röcker, Carlheinz; Nienhaus, G. Ulrich; Möller, Martin

doi:10.1021/ja0318028

Cited by 183 publications

(182 citation statements)

References 41 publications

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“…We have recently presented a method of protein immobilization on a glass surface that ensures minimal interaction with the surface in both the native and the GdmCldenatured states (19,20). Briefly, glass coverslips were coated with star-shaped PEG molecules containing reactive isocyanate end groups (SusTech, Darmstadt, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, we developed an alternative strategy of immobilization, in which the protein molecules are attached to a glass surface densely coated with poly(ethylene glycol) (PEG) star-shaped polymers through a streptavidin-biotin linkage (19,20). Of key importance for protein-folding studies is the observation that interactions between the surface and both the folded and unfolded protein molecules are negligible, as judged from the free energy change, ⌬G, the cooperativity parameter m of folding, and essentially complete unfolding-refolding reversibility (19,20).Here, we present a single-molecule study of the dynamics of a small enzyme, ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl). RNase H was chosen because it is a model system for protein folding, well-characterized by equilibrium and kinetic experiments including mutational analysis (21-23).…”

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confidence: 99%

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Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

Kuzmenkina

Heyes

Nienhaus

2005

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

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173

172

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Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfoldedstate ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Fö rster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of Ϸ20 s at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of Ϸ2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.fluorescence resonance energy transfer ͉ guanidinium chloride ͉ protein folding ͉ RNase H ͉ single-molecule spectroscopy P roteins are complex systems that can exist in a huge number of different conformations. Even in the properly folded, native state, the polypeptide chain adopts many slightly different conformations that can be depicted as local minima in a rugged energy landscape, and relaxations and fluctuations in the landscape are crucially involved in functional processes (1, 2). Conformational heterogeneity is even more of a concern in studies of protein-folding reactions because of the vast number of possible arrangements of the unfolded polypeptide chain and the many complex pathways leading from the ensemble of unfolded to the native conformations in an overall funnel-shaped energy landscape (3-7).Because of Anfinsen's key observation that the native fold is already encoded in the sequence of amino acids (8), the proteinfolding problem has attracted enormous attention, and the field has progressed in a healthy interplay between theory and experiment. A wide variety of biophysical approaches were developed to follow the dynamics of the polypeptide en route to the native state. Valuable insights have been achieved by timeresolved spectroscopic experiments on bu...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

Kuzmenkina

Heyes

Nienhaus

2005

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

Self Cite

173

172

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“…As for Csp, sequential labeling was employed to equip RNAse H with Alexa Fluor 546 and Alexa Fluor 647 fluorophores at two engineered cysteines, but in contrast to the burst spectroscopy studies discussed above, Nienhaus and colleagues opted for single-molecule experiments with immobilized protein. 170,171 To prevent unspecific surface adsorption of the immobilized protein previously seen in another study, 127,139 the authors used chemically designed, biotinylated surface coatings prepared by spin coating (Figure 10). The surface coatings were made of ultrathin networks of isocyanate-terminated "star-shaped" poly(ethylene oxide) (PEO) molecules, cross-linked at their ends via urea groups to minimize the intertwining of a denatured polypeptide chain with the PEO polymer.…”

Section: Ribonuclease H (Rnase H)-rnasementioning

confidence: 99%

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Michalet¹,

Weiss²,

Jaeger³

2006

ChemInform

125

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“…Additionally, they must have a negligible impact on the dynamics of the attached proteins. Nienhaus and coworkers (9,10) have developed and analyzed several types of surfaces, deemed minimally interacting, for the explicit purpose of performing experiments on individually tethered biomolecules. In particular, they have designed a star-shaped polyethylene glycol (PEG) surface that allows for the reversible folding and unfolding of attached proteins (10,11).…”

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confidence: 99%

Effects of surface tethering on protein folding mechanisms

Friedel¹,

Baumketner

Shea

2006

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

113

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The folding mechanisms of proteins are increasingly being probed through single-molecule experiments in which the protein is immobilized on a surface. Nevertheless, a clear understanding of how the surface might affect folding, and whether or not it changes folding from its bulk behavior, is lacking. In this work, we use molecular dynamics simulations of a model ␤-barrel protein tethered to a surface to systematically investigate how the surface impacts folding. In the bulk, this protein folds in a three-state manner through a compact intermediate state, and its transition state (TS) has a well formed hydrophobic core. Upon tethering, we find that folding rates and stability are impacted differently by the surface, with dependencies on both the length and location of the tether. Significant changes in folding times are observed for tether points that do not alter the folding temperature. Tethering also locally enhances the formation of structure for residues proximal to the tether point. We find that neither the folding mechanism nor the TS of this protein are altered if the tether is in a fully structured or completely unstructured region of the TS. By contrast, tethering in a partially structured region of the TS leads to dramatic changes. For one such tether point, the intermediate present in bulk folding is eliminated, leading to a two-state folding process with a heterogeneous, highly unstructured TS ensemble. These results have implications for both the design of single-molecule experiments and biotechnological applications of tethered proteins. molecular dynamics simulations ͉ protein-surface interactions ͉ single-molecule spectroscopy S ingle-molecule spectroscopy has recently emerged as a powerful technique for watching individual proteins fold (1-3). By attaching donor and acceptor dyes to key residues of a protein, fluorescence resonance energy transfer (FRET), already successful in ensemble folding experiments (4), can be used as a distance probe to monitor individual folding pathways. There are several different experimental techniques for doing FRET-based single-molecule experiments on proteins, each with distinct advantages and challenges. Bulk experiments use a focusing laser beam that monitors folding as proteins diffuse freely through the area illuminated by the laser (5, 6). Although this approach is advantageous in that the protein is allowed to fold in a relatively nondisrupted manner, solution experiments are diffusion limited and cannot examine slower (տ10 ms) phenomena (2). Proteins enclosed in surface-tethered vesicles allow observations on a more spatially localized scale than the bulk but do not allow for the rapid exchange of buffer conditions or the use of extreme denaturing environments (7). By immobilizing them directly on a surface (8), proteins can be observed in both a spatially localized region and over longer time scales than those accessible in diffusion-limited experiments. Despite these advantages, the folding behavior of surface-tethered proteins also may be influenced by ...

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Biofunctionalized, Ultrathin Coatings of Cross-Linked Star-Shaped Poly(ethylene oxide) Allow Reversible Folding of Immobilized Proteins

Cited by 183 publications

References 41 publications

Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Effects of surface tethering on protein folding mechanisms

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