From Protein Design to the Energy Landscape of a Cold Unfolding Protein

Pulavarti, S.; Maguire, Jack; Yuen, Shirley; Harrison, Joseph S.; Griffin, Jermel; Premkumar, Lakshmanane; Esposito, Edward A.; Makhatadze, George I.; Garcı́a, Angel E.; Weiß, Thomas; Snell, Edward H.; Kuhlman, Brian; Szyperski, Thomas

doi:10.1021/acs.jpcb.1c10750

Cited by 4 publications

(3 citation statements)

References 110 publications

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“…The protein concentration was set at 5 μM in 2 mL of solution in a buffer of 20 mM Na 2 HPO 4 /NaH 2 PO 4 (pH 7.0) with 50 mM NaCl. In addition to the emission spectra, the center of spectral mass (CM) was calculated using eq : , CM = ∑ i λ i F i ∑ i F i where F i is the emission in the wavelength λ i .…”

Section: Methodsmentioning

confidence: 99%

Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding

Dias,

Pedro,

Sanches

et al. 2023

J. Chem. Inf. Model.

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The folding process of multidomain proteins is a highly intricate phenomenon involving the assembly of distinct domains into a functional three-dimensional structure. During this process, each domain may fold independently while interacting with others. The folding of multidomain proteins can be influenced by various factors, including their composition, the structure of each domain, or the presence of disordered regions, as well as the surrounding environment. Misfolding of multidomain proteins can lead to the formation of nonfunctional structures associated with a range of diseases, including cancers or neurodegenerative disorders. Understanding this process is an important step for many biophysical analyses such as stability, interaction, malfunctioning, and rational drug design. One such multidomain protein is growth factor receptor-bound protein 2 (GRB2), an adaptor protein that is essential in regulating cell survival. GRB2 consists of one central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. The SH2 domain interacts with phosphotyrosine regions in other proteins, while the SH3 domains recognize proline-rich regions on protein partners during cell signaling. Here, we combined computational and experimental techniques to investigate the folding process of GRB2. Through computational simulations, we sampled the conformational space and mapped the mechanisms involved by the free energy profiles, which may indicate possible intermediate states. From the molecular dynamics trajectories, we used the energy landscape visualization method (ELViM), which allowed us to visualize a three-dimensional (3D) representation of the overall energy surface. We identified two possible parallel folding routes that cannot be seen in a onedimensional analysis, with one occurring more frequently during folding. Supporting these results, we used differential scanning calorimetry (DSC) and fluorescence spectroscopy techniques to confirm these intermediate states in vitro. Finally, we analyzed the deletion of domains to compare our model outputs to previously published results, supporting the presence of interdomain modulation. Overall, our study highlights the significance of interdomain communication within the GRB2 protein and its impact on the formation, stability, and structural plasticity of the protein, which are crucial for its interaction with other proteins in key signaling pathways.

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Section: Methodsmentioning

confidence: 99%

Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding

Dias,

Pedro,

Sanches

et al. 2023

J. Chem. Inf. Model.

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“…We have recently shown that protein design is poised to play a key role to explore protein cold unfolding energy landscapes, the mechanisms of cold unfolding, and to robustly test related theories . Specifically, we designed cold unfolding 4-helix bundles with de novo designed bipartite hydrophilic/hydrophobic cores featuring hydrogen-bond networks to study the relative importance of hydrophobic versus hydrophilic protein–water interactions for cold unfolding.…”

Section: Discussionmentioning

confidence: 99%

“…9 We have recently shown that protein design is poised to play a key role to explore protein cold unfolding energy landscapes, the mechanisms of cold unfolding, and to robustly test related theories. 38 Specifically, we designed cold unfolding 4-helix bundles with de novo designed bipartite hydrophilic/hydrophobic cores featuring hydrogen-bond networks to study the relative importance of hydrophobic versus hydrophilic protein−water interactions for cold unfolding. Our strategy presented here to design inside-out zinc-binding sites offers new opportunities for the design of cold unfolding proteins with zinc coordination sites representing the hydrophilic segment that potentially accommodates intruding waters during the onset of cold unfolding.…”

Section: ■ Discussionmentioning

confidence: 99%

Inside-Out Design of Zinc-Binding Proteins with Non-Native Backbones

et al. 2023

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The de novo design of functional proteins requires specification of tertiary structure and incorporation of molecular binding sites. Here, we develop an inside-out design strategy in the molecular modeling program Rosetta that begins with amino acid side chains from one or two α-helices making well-defined contacts with a ligand. A full-sized protein is then built around the ligand by adding additional helices that promote the formation of a protein core and allow additional contacts with the ligand. The protocol was tested by designing 12 zinc-binding proteins, each with 4−5 helices. Four of the designs were folded and bound to zinc with equilibrium dissociation constants varying between 95 nM and 1.1 μM. The design with the tightest affinity for zinc, N12, adopts a unique conformation in the folded state as assessed with nuclear magnetic resonance (NMR) and the design model closely matches (backbone root-mean-square deviation (RMSD) < 1 Å) an AlphaFold model of the sequence. Retrospective analysis with AlphaFold suggests that the sequences of many of the failed designs did not encode the desired tertiary packing.

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Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence

Igwe,

Müller,

Gisperg

et al. 2024

Anal Bioanal Chem

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Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L$$^{-1}$$ - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding. Graphical abstract Real-time intrinsic fluorescence monitoring in protein refolding

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From Protein Design to the Energy Landscape of a Cold Unfolding Protein

Cited by 4 publications

References 110 publications

Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding

Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding

Inside-Out Design of Zinc-Binding Proteins with Non-Native Backbones

Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence

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