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Gary J Pielak - One of the best experts on this subject based on the ideXlab platform.
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residue level quantification of Protein Stability in living cells
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: William B Monteith, Gary J PielakAbstract:The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic Protein Stability under nonperturbing conditions. We report the use of NMR-detected hydrogen–deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-aa B1 domain of Protein G (GB1) in living Escherichia coli cells without adding destabilizing cosolutes or heat. Comparisons to dilute solution data (pH 7.6 and 37 °C) show that opening free energies increase by as much as 1.14 ± 0.05 kcal/mol in cells. Importantly, we also show that homogeneous Protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. We discuss our findings in terms of hard-core excluded volume effects, charge–charge GB1-crowder interactions, and other factors. The quenched lysate method identifies the residues most important for folding GB1 in cells, and should prove useful for quantifying the Stability of other globular Proteins in cells to gain a more complete understanding of the effects of the intracellular environment on Protein chemistry.
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impact of reconstituted cytosol on Protein Stability
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Mohona Sarkar, Austin E Smith, Gary J PielakAbstract:Protein Stability is usually studied in simple buffered solutions, but most Proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMR-detected amide proton exchange was then used to quantify the Stability of the globular Protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous Protein crowders, reinforcing the biological significance of weak, nonspecific interactions.
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macromolecular crowding and Protein Stability
Journal of the American Chemical Society, 2012Co-Authors: Yaqiang Wang, Mohona Sarkar, Austin E Smith, Alexander S Krois, Gary J PielakAbstract:An understanding of cellular chemistry requires knowledge of how crowded environments affect Proteins. The influence of crowding on Protein Stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on Protein Stability, however, focus on hard-core repulsions, which are inherently entropic and stabilizing. We assessed these phenomena by measuring the temperature dependence of NMR-detected amide proton exchange and used these data to extract the entropic and enthalpic contributions of crowding to the Stability of ubiquitin. Contrary to expectations, the contribution of chemical interactions is large and in many cases dominates the contribution from hardcore repulsions. Our results show that both chemical interactions and hard-core repulsions must be considered when assessing the effects of crowding and help explain previous observations about Protein Stability and dynamics in cells.
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volume exclusion and soft interaction effects on Protein Stability under crowded conditions
Biochemistry, 2010Co-Authors: Andrew C Miklos, Naima G Sharaf, Gary J PielakAbstract:Most Proteins function in nature under crowded conditions, and crowding can change Protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams of macromolecules per liter often interfere with the observation of the Protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test Protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium Stability of a globular Protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test Protein. Stability was quantified with NMR-detected amide 1H exchange. We analyzed the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a ...
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using nmr detected backbone amide 1h exchange to assess macromolecular crowding effects on globular Protein Stability
Methods in Enzymology, 2009Co-Authors: Andrew C Miklos, Conggang Li, Gary J PielakAbstract:The biophysical properties of Proteins in the crowded intracellular environment are expected to differ from those for Proteins in dilute solution. Crowding can be studied in vitro through addition of polymers at high concentrations. NMR-detected amide 1H exchange is the only technique that provides equilibrium Stability data for Proteins on a per-residue basis under crowded conditions. We describe the theory behind amide 1H exchange and provide a detailed description of the experiments used to quantify globular Protein Stability at the residue level under crowded conditions. We also discuss the detection of weak interactions between the test Protein and the crowding molecules.
Stephen L Mayo - One of the best experts on this subject based on the ideXlab platform.
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Protein Stability engineering insights revealed by domain wide comprehensive mutagenesis
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Alex Nisthal, Connie Y Wang, Marie L Ary, Stephen L MayoAbstract:The accurate prediction of Protein Stability upon sequence mutation is an important but unsolved challenge in Protein engineering. Large mutational datasets are required to train computational predictors, but traditional methods for collecting Stability data are either low-throughput or measure Protein Stability indirectly. Here, we develop an automated method to generate thermodynamic Stability data for nearly every single mutant in a small 56-residue Protein. Analysis reveals that most single mutants have a neutral effect on Stability, mutational sensitivity is largely governed by residue burial, and unexpectedly, hydrophobics are the best tolerated amino acid type. Correlating the output of various Stability-prediction algorithms against our data shows that nearly all perform better on boundary and surface positions than for those in the core and are better at predicting large-to-small mutations than small-to-large ones. We show that the most stable variants in the single-mutant landscape are better identified using combinations of 2 prediction algorithms and including more algorithms can provide diminishing returns. In most cases, poor in silico predictions were tied to compositional differences between the data being analyzed and the datasets used to train the algorithm. Finally, we find that strategies to extract stabilities from high-throughput fitness data such as deep mutational scanning are promising and that data produced by these methods may be applicable toward training future Stability-prediction tools.
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Protein Stability engineering insights revealed by domain wide comprehensive mutagenesis
bioRxiv, 2018Co-Authors: Alex Nisthal, Connie Y Wang, Marie L Ary, Stephen L MayoAbstract:Abstract The accurate prediction of Protein Stability upon sequence mutation is an important but unsolved challenge in Protein engineering. Large mutational datasets are required to train computational predictors, but traditional methods for collecting Stability data are either low-throughput or measure Protein Stability indirectly. Here, we develop an automated method to generate thermodynamic Stability data for nearly every single mutant in a small 56-residue Protein. Analysis reveals that most single mutants have a neutral effect on Stability, mutational sensitivity is largely governed by residue burial, and unexpectedly, hydrophobics are the best tolerated amino acid type. Correlating the output of various Stability prediction algorithms against our data shows that nearly all perform better on boundary and surface positions than for those in the core, and are better at predicting large to small mutations than small to large ones. We show that the most stable variants in the single mutant landscape are better identified using combinations of two prediction algorithms, and that including more algorithms can provide diminishing returns. In most cases, poor in silico predictions were tied to compositional differences between the data being analyzed and the datasets used to train the algorithm. Finally, we find that strategies to extract stabilities from high-throughput fitness data such as deep mutational scanning are promising and that data produced by these methods may be applicable toward training future Stability prediction tools. Significance Statement Using liquid-handling automation, we constructed and measured the thermodynamic Stability of almost every single mutant of Protein G (Gβ1), a small domain. This self-consistent dataset is the largest of its kind and offers unique opportunities on two fronts: (i) insight into Protein domain properties such as positional sensitivity and incorporated amino acid tolerance, and (ii) service as a validation set for future efforts in Protein Stability prediction. As Gβ1 is a model system for Protein folding and design, and its single mutant landscape has been measured by deep mutational scanning, we expect our dataset to serve as a reference for studies aimed at extracting Stability information from fitness data or developing novel high-throughput Stability assays.
Chiwook Park - One of the best experts on this subject based on the ideXlab platform.
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determining Protein Stability in cell lysates by pulse proteolysis and western blotting
Protein Science, 2009Co-Authors: Moonsoo Kim, Jiao Song, Chiwook ParkAbstract:Proteins require proper conformational energetics to fold and to function correctly. Despite the importance of having information on conformational energetics, the investigation of thermodynamic Stability has been limited to Proteins, which can be easily expressed and purified. Many biologically important Proteins are not suitable for conventional biophysical investigation because of the difficulty of expression and purification. As an effort to overcome this limitation, we have developed a method to determine the thermodynamic Stability of low abundant Proteins in cell lysates. Previously, it was demonstrated that Protein Stability can be determined quantitatively by measuring the fraction of folded Proteins with a pulse of proteolysis (Pulse proteolysis). Here, we show that thermodynamic Stability of low abundant Proteins can be determined reliably in cell lysates by combining pulse proteolysis with quantitative Western blotting (Pulse and Western). To demonstrate the reliability of this method, we determined the thermodynamic Stability of recombinant human H-ras added to lysates of E. coli and human Jurkat T cells. Comparison with the thermodynamic Stability determined with pure H-ras revealed that Pulse and Western is a reliable way to monitor Protein Stability in cell lysates and the Stability of H-ras is not affected by other Proteins present in cell lysates. This method allows the investigation of conformational energetics of Proteins in cell lysates without cloning, purification, or labeling.
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pulse proteolysis a simple method for quantitative determination of Protein Stability and ligand binding
Nature Methods, 2005Co-Authors: Chiwook Park, Susan MarquseeAbstract:Thermodynamic Stability is fundamental to the biology of Proteins. Information on Protein Stability is essential for studying Protein structure and folding and can also be used indirectly to monitor Protein-ligand or Protein-Protein interactions. While clearly valuable, the experimental determination of a Protein's Stability typically requires biophysical instrumentation and substantial quantities of purified Protein, which has limited the use of this technique as a general laboratory method. We report here a simple new method for determining Protein Stability by using pulse proteolysis with varying concentrations of denaturant. Pulse proteolysis is designed to digest only the unfolded Proteins in an equilibrium mixture of folded and unfolded Proteins that relaxes on a time scale longer than the proteolytic pulse. We used this method to study the stabilities of Escherichia coli ribonuclease H and its variants, both in purified form and directly from cell lysates. The ΔGunf° values obtained by this technique were in agreement with those determined by traditional methods. We also successfully used this method to monitor the binding of maltose-binding Protein to maltose, as well as to rapidly screen cognate ligands for this Protein. The simplicity of pulse proteolysis suggests that it is an excellent strategy for the high-throughput determination of Protein Stability in Protein engineering and drug discovery applications.
Xu Cao - One of the best experts on this subject based on the ideXlab platform.
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smad4 Protein Stability is regulated by ubiquitin ligase scfβ trcp1
Journal of Biological Chemistry, 2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming Shi, Xu CaoAbstract:Smad4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein Stability is unclear. We have identified SCFβ-TrCP1, a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein β-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The β-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing β-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous β-TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-β-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCFβ-TrCP1 abrogates TGF-β function in vivo by decreasing Smad4 Stability.
Mei Wan - One of the best experts on this subject based on the ideXlab platform.
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smad4 Protein Stability is regulated by ubiquitin ligase scfβ trcp1
Journal of Biological Chemistry, 2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming Shi, Xu CaoAbstract:Smad4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein Stability is unclear. We have identified SCFβ-TrCP1, a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein β-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The β-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing β-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous β-TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-β-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCFβ-TrCP1 abrogates TGF-β function in vivo by decreasing Smad4 Stability.
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smad4 Protein Stability is regulated by ubiquitin ligase scf
2004Co-Authors: Mei Wan, Yi Tang, Ewan M Tytler, Bingwen Jin, Selwyn M Vickers, Lei Yang, Xing Ming ShiAbstract:Smad4 is a key intracellular mediator for the transforming growth factor(TGF) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) Proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 Protein Stability is unclear. We have identified SCF , a ubiquitin (E3) ligase, as a critical determinant for the Protein degradation of Smad4 Protein. F-box Protein -TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The -TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing -TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous -TrCP1 suppression increases the expression of Smad4 Protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCF -TrCP1 abrogates TGFfunction in vivo by decreasing Smad4 Stability.
