Mechanical Stability Of Proteins Research Articles

Role of Nonspecific Interactions in Molecular Chaperones through Model-Based Bioinformatics

Molecular chaperones are large proteins or protein complexes from which many proteins require assistance in order to fold. One unique property of molecular chaperones is the cavity they provide in which proteins fold. The interior surface residues which make up the cavities of molecular chaperone complexes from different organisms has recently been identified, including the well-studied GroEL-GroES chaperonin complex found in E. coli. It was found that the interior of these protein complexes is significantly different than other protein surfaces and that the residues found on the protein surface are able to resist protein adsorption when immobilized on a surface. Yet it remains unknown if these residues passively resist protein binding inside GroEL-GroEs, as demonstrated by experiments which created synthetic mimics of the interior cavity, or if the interior also actively stabilizes protein folding. To answer this question, we have extended entropic models of substrate protein folding inside GroEL-GroES to include interaction energies between substrate proteins and the GroEL-GroES chaperone complex. This model was tested on a set of 528 proteins and the results qualitatively match experimental observations. The interior residues were found to strongly discourage the exposure of any hydrophobic residues, providing an enhanced hydrophobic effect inside the cavity which actively influences protein folding. This work provides both a mechanism for active protein stabilization in GroEL-GroES and a model which matches current understanding the chaperone protein.

Histone Demethylase Jumonji D3 (JMJD3) as a Tumor Suppressor by Regulating p53 Protein Nuclear Stabilization

Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3) resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs), where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation) and chromatin independent (nuclear p53 protein stabilization) mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.

Structure and effect of sarcosine on water and urea by using molecular dynamics simulations: Implications in protein stabilization

Sarcosine is one of the most important protecting osmolytes which is also known to counteract the denaturing effect of urea. We used molecular dynamics simulation methods to investigate the mechanism of protein stabilization and counteraction of urea by sarcosine. We found that sarcosine enhanced the tetrahedral structure of water and strengthened its hydrogen bonding network. We also found that sarcosine did not form clusters unlike glycine. Our results show strong interaction between sarcosine and urea molecules. Addition of sarcosine enhanced the urea–water structure and urea–water lifetime indicated an increase in the solvation of urea. These findings suggest that sarcosine indirectly stabilizes protein by enhancing water–water structure thus decreasing the hydrophobic effect and counteracts the effect of urea by increasing the solvation of urea and directly interacting with it leaving urea less available to interact with protein.

Role of Nonspecific Interactions in Molecular Chaperones through Model-Based Bioinformatics

Molecular chaperones are large proteins or protein complexes from which many proteins require assistance in order to fold. One unique property of molecular chaperones is the cavity they provide in which proteins fold. The interior surface residues which make up the cavities of molecular chaperone complexes from different organisms has recently been identified, including the well-studied GroEL-GroES chaperonin complex found in Escherichia coli. It was found that the interior of these protein complexes is significantly different than other protein surfaces and that the residues found on the protein surface are able to resist protein adsorption when immobilized on a surface. Yet it remains unknown if these residues passively resist protein binding inside GroEL-GroEs (as demonstrated by experiments that created synthetic mimics of the interior cavity) or if the interior also actively stabilizes protein folding. To answer this question, we have extended entropic models of substrate protein folding inside GroEL-GroES to include interaction energies between substrate proteins and the GroEL-GroES chaperone complex. This model was tested on a set of 528 proteins and the results qualitatively match experimental observations. The interior residues were found to strongly discourage the exposure of any hydrophobic residues, providing an enhanced hydrophobic effect inside the cavity that actively influences protein folding. This work provides both a mechanism for active protein stabilization in GroEL-GroES and a model that matches contemporary understanding of the chaperone protein.

Single Molecule Force Spectroscopy Reveals Critical Roles of Hydrophobic Core Packing in Determining the Mechanical Stability of Protein GB1

Understanding molecular determinants of protein mechanical stability is important not only for elucidating how elastomeric proteins are designed and functioning in biological systems but also for designing protein building blocks with defined nanomechanical properties for constructing novel biomaterials. GB1 is a small α/β protein and exhibits significant mechanical stability. It is thought that the shear topology of GB1 plays an important role in determining its mechanical stability. Here, we combine single molecule atomic force microscopy and protein engineering techniques to investigate the effect of side chain reduction and hydrophobic core packing on the mechanical stability of GB1. We engineered seven point mutants and carried out mechanical φ-value analysis of the mechanical unfolding of GB1. We found that three mutations, which are across the surfaces of two subdomains that are to be sheared by the applied stretching force, in the hydrophobic core (F30L, Y45L, and F52L) result in significant decrease in mechanical unfolding force of GB1. The mechanical unfolding force of these mutants drop by 50-90 pN compared with wild-type GB1, which unfolds at around 180 pN at a pulling speed of 400 nm/s. These results indicate that hydrophobic core packing plays an important role in determining the mechanical stability of GB1 and suggest that optimizing hydrophobic interactions across the surfaces that are to be sheared will likely be an efficient method to enhance the mechanical stability of GB1 and GB1 homologues.

Abstract LB-209: Identification and characterization of small molecule inhibitor of Wnt canonical pathway in a glioma setting.

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors. The aggressive and highly invasive phenotype of these tumors makes them among the more destructive human cancers with a median survival of less than one year. In the different GBM tumor subtypes several altered genes and multiple pathways cooperate to promote and sustain tumor growth, tumor invasion and tumor recurrence. Although Wnt pathway activation was historically linked to the presence of mutations involving key components of the network (APC, β-catenin or AXIN proteins), an increasing number of studies suggest that aberrant Wnt signaling can also be initiated by several alternative mechanisms. Autocrine signaling mediated by specific Wnt ligands has been linked to lung, breast and pancreatic tumours, but also malignant melanoma cells spreading. Wnt signals, both positive and negative, form a class of paracrine growth factors that could act to influence multiple myeloma cell growth, metastatic potential and target tissue erosion. Although very well studied in multiple diseases, the role and importance of Wnt signaling pathway has not been extensively described in GBM tumors. After an initial phase where we showed modulation of the Wnt transcriptional activity and the phenotypic consequences of negative Wnt signaling after β-catenin KO in glioma cells we started a screening campaign to identify small molecules Wnt inhibitors coupling a pathway/phenotypic approach to oncology relevant phenotypic assays. Among the different chemical classes identified, we characterized a selective canonical Wnt signaling inhibitor, which stabilizes axin (a negative regulator of the Wnt signalling pathway) at the protein level together with a concomitant decrease at the transcriptional level. Due to the central role of axin in controlling the ratio between the unphosphorylated (the stable form which can then enter the nucleus and thus activate Wnt target genes) and the phosphorylated (labelled for proteasomal degradation) pool of β-catenin we also observed as a consequence an increase in the amount of cytosolic phosphorylated β-catenin (S33/S37/T41) and a decrease of total β-catenin. One possible explanation for the up-regulation of axin protein level and the concomitant decrease of steady-state axin mRNA levels after compound treatment could be via a protein stabilization mechanism, as demonstrated by the effects of the small molecule on the half-life of axin2 in DBTRG cells. Consistent with a protective effect of the molecule against axin2 proteasomal degradation, co-treatment of the Wnt inhibitor and the reversible proteasome inhibitor MG-132 almost completely blocked the ubiquitination of axin2. In vivo studies, used to confirm the in vitro observations, showed antitumor activity in a glioma xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-209. doi:1538-7445.AM2012-LB-209

The effect of functionalization of mesoporous silica nanoparticles on the interaction and stability of confined enzyme

Immobilization of enzymes into the mesoporous nanomaterials results in formation of more stable and even more active versions of biocatalysts. The effect of surface functionalization of mesoporous silica nanoparticles (MSNs) on its adsorption characteristics and stability of superoxide dismutase (SOD) was investigated. For this purpose, non-functionalized (KIT-6) and aminopropyl-functionalized cubic Ia3d mesoporous silica ([n-PrNH2-KIT-6]) nanoparticles with 3-dimensional pores were used as supports. It was observed that the amount of enzyme adsorbed on/within MSNs is dependent on the initial enzyme concentration for both KIT-6 and [n-PrNH2-KIT-6] mesoporous silicas. However a stronger interaction between SOD and [n-PrNH2-KIT-6] was observed relative to KIT-6. Increasing temperature favors a larger amount of SOD immobilization into KIT-6, while it was negligible for [n-PrNH2-KIT-6]. Immobilized SOD was more stable against urea and thermal denaturation relative to free enzyme and this improvement of stability was more pronounced for SOD into the [n-PrNH2-KIT-6] than KIT-6. These results may be useful in determining the mechanism(s) of protein immobilization and stabilization into the solid supports.

Mapping Site-Specific Changes That Affect Stability of the N-Terminal Domain of Calmodulin

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability.

Protein Hydration and Excluded Volume Interactions in Protein Folding and Stability: On the Mechanism of Protein Stabilization by Osmolytes

Several osmolytes ranging in size from ∼90-1600 cm3.mol-1 are shown to stabilize the folded states of apo-Mb at pH denaturing conditions. The action of these solutes is consistent with an osmotic stress effect, i.e., with their effect on the chemical potential of water modulating the equilibrium between folded and unfolded states via protein differential hydration. Moreover, the free energy of protein folding depends on solute size. We show that the apparent difference in hydration between molten globule and unfolded states of apo-Mb increases linearly with increasing solute sizes, while the free energy change of protein hydration upon folding decreases with the inverse of molar solute apparent volume. We have analyzed these size effects considering the contribution of excluded volume interactions to protein folding via Monte Carlo simulations of a self-avoiding walk chain in a cubic lattice with hardcore solutes. It is shown that solute-chain self-avoiding interaction decrease the conformational entropy of the chain in proportion to its square radius of gyration, and to the solute volume fraction occupied by the solute. The computational results translate into a difference in the chemical potential between folded and unfolded protein states that remarkably predicts the experimental influence of solute sizes and water chemical potential on the free energy change of apo-Mb refolding induced by osmolytes. Thus, this work may stablish a quantitative link between protein hydration and protein excluded-volume interactions and their effect on the energetic of protein folding.

Enhancing the Mechanical Stability of Proteins through a Cocktail Approach

Rationally enhancing the mechanical stability of proteins remains a challenge in the field of single molecule force spectroscopy. Several strategies have been developed successfully to rationally regulate the mechanical stability of proteins. These strategies include rational control of the unfolding pathway by disulfide bond formation, improving hydrophobic packing, reconstruction of the force-bearing region of proteins, ligand binding, and engineered metal chelation. However, compared with the well-developed methods used to enhance the thermodynamic stability of proteins/enzymes, these methods to enhance the mechanical stability remain limited. Furthermore, these methods are only used one at a time, and the resulted enhancement in protein mechanical stability is also rather limited. Possible synergetic effects from more than one method remain largely unexplored. Here we use single molecule force spectroscopy techniques to demonstrate that it is feasible to use a ‘‘cocktail’’ approach for combining more than one approach to enhance significantly the mechanical stability of proteins in an additive fashion. As a proof of principle, we show that metal chelation and protein-protein interaction can be combined to enhance the unfolding force of a protein to ∼450 pN, which is >3 times of its original value. This is also higher than the mechanical stability of most of proteins studied so far. We also extend such a cocktail concept to combine two different metal chelation sites to enhance protein mechanical stability. This approach opens new avenues to efficiently regulating the mechanical properties of proteins, and should be applicable to a wide range of elastomeric proteins.

Enhancing the Mechanical Stability of Proteins through a Cocktail Approach

Rationally enhancing the mechanical stability of proteins remains a challenge in the field of single molecule force spectroscopy. Here we demonstrate that it is feasible to use a “cocktail” approach for combining more than one approach to enhance significantly the mechanical stability of proteins in an additive fashion. As a proof of principle, we show that metal chelation and protein-protein interaction can be combined to enhance the unfolding force of a protein to ∼450 pN, which is >3 times of its original value. This is also higher than the mechanical stability of most of proteins studied so far. We also extend such a cocktail concept to combine two different metal chelation sites to enhance protein mechanical stability. This approach opens new avenues to efficiently regulating the mechanical properties of proteins, and should be applicable to a wide range of elastomeric proteins.

Widespread Disulfide Bonding in Proteins from Thermophilic Archaea

Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaeal branch, which are essentially all hyperthermophilic, are universally rich in disulfide bonding while lesser degrees of disulfide bonding are found among the thermophilic Euryarchaea, excluding those that are methanogenic. The results help clarify which parts of the archaeal lineage are likely to yield more examples and additional specific data on protein disulfide bonding, as increasing genomic sequencing efforts are brought to bear.

Compacting Proteins: Pros and Cons of Osmolyte-Induced Folding

Biomedical applications of osmolytes, including stabilization of protein-based pharmaceutics, preservation of living biological material and potential therapeutic prescription in vivo, are intimately related to the fact that osmolytes favour the native structure of proteins. The shift towards the native structure is associated to the compaction of the protein by a non-specific mechanism. This compaction is observed mostly for the unfolded state but also for the transition state ensemble and even for the native state. In addition, more stable three-dimensional structures are more stabilized by osmolytes if the overall protein fold is the same indicating that point mutations and osmolytes should share a similar mechanism for protein stabilization. A synergistic effect to increase protein stability between accumulation of osmolytes and protein engineering strategies seems to have operated during evolution. However, the conformational pre-organization of the unfolded state (compaction) induced by osmolytes which increases the folding rate, might lead to the accumulation of off-folding pathway intermediates with non-native structure that delay folding. Also, osmolytes favor protein aggregation as an alternative way to shield protein surfaces from the solvent. The sometimes observed effect of osmolytes on the prevention of protein aggregation is apparent as they only decrease the accumulation of aggregation-competent partially unfolded states.

Hypoxia-inducible Factor-1 Activation in Nonhypoxic Conditions: The Essential Role of Mitochondrial-derived Reactive Oxygen Species

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1α transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22(phox) siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1α protein stability, HIF-1-dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase-generated ROS in Ang II-mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions.

In Silico Structural and Functional Analysis of Fragments of the Ankyrin Repeat Protein p18INK4c

Ankyrin repeat proteins (ARPs) are ubiquitous proteins that play critical regulatory roles in organisms and consist of repeating motifs (ankyrin repeats) stacked in non-globular, almost linear, “quasi one-dimensional” configurations. They also have highly unusual mechanical properties, notably ARPs can behave as nano-springs. Both their essential cellular functions and distinctive nano-mechanical properties have aroused interest in ARPs for potential applications in medicine and nanotechnology. Further, the modular architecture of ARPs, which lack the long-range contacts that typically stabilize globular proteins, provides a new paradigm for understanding protein stability and folding mechanisms of proteins. In the present study, the stability of ARP p18INK4c (p18) and fifty p18 fragments was investigated by all-atomic molecular dynamics (MD) simulations in explicit water on a ∼3.3 microseconds timescale. The fragment simulations indicate that p18 a-helices are significantly stabilized by tertiary interactions, because in the absence of their native context they readily melt. All single p18 ARs and their structural elements are also unstable outside their native context. The minimal stable motifs are pairs of ARs, implying that inter-repeat contacts are essential for AR stability. Further, pairs of internal ARs are less stable than pairs that include a native capping AR. The MD simulations also provide indications of the functional roles of p18 turns and loops; the turns appear to be essential for the stability of the protein, while the loops both help to stabilize the p18 structure and are involved in recognition processes. Temperature-induced unfolding analysis shows that the p18 melts from the N-terminus to the C-terminus.

Smad3 Prevents β-Catenin Degradation and Facilitates β-Catenin Nuclear Translocation in Chondrocytes

Our previous study demonstrated that transforming growth factor (TGF)-beta activates beta-catenin signaling through Smad3 interaction with beta-catenin in chondrocytes. In the present studies, we further investigated the detailed molecular mechanism of the cross-talk between TGF-beta/Smad3 and Wnt/beta-catenin signaling pathways. We found that C-terminal Smad3 interacted with both the N-terminal region and the middle region of beta-catenin protein in a TGF-beta-dependent manner. Both Smad3 and Smad4 were required for the interaction with beta-catenin and protected beta-catenin from an ubiquitin-proteasome-dependent degradation. In addition, the formation of the Smad3-Smad4-beta-catenin protein complex also mediated beta-catenin nuclear translocation. This Smad3-mediated regulatory mechanism of beta-catenin protein stability enhanced the activity of beta-catenin to activate downstream target genes during chondrogenesis. Our findings demonstrate a novel mechanism between TGF-beta and Wnt/beta-catenin signaling pathways during chondrocyte development.

ChemInform Abstract: Mechanisms of Protein Stabilization in the Solid State

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Mechanisms of Protein Stabilization and Prevention of Protein Aggregation by Glycerol

The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices

Biopharmaceutical proteins are often formulated and freeze dried in agents that protect them from deleterious reactions that can compromise activity and authenticity. Although such approaches are widely used, a detailed understanding of the molecular mechanisms of protein stabilization in low water content amorphous glasses is lacking. Further, whilst deterioration chemistries are well described in dilute solution, relatively little is known about the extent and mechanisms by which protein integrity is compromised in the glassy state. Here we have investigated the relationship between protein modification and rate thereof, with variation of pH, carbohydrate excipient, temperature and the glass transition temperature using a model protein, lysozyme. Mass spectrometry analysis and peptide mapping confirm that protein modifications do occur in the glassy state in a time-, temperature-, and carbohydrate excipient-dependent manner. There were clear trends between the buffer pH and the primary modification detected (glycation). Most importantly, there were differences in the apparent reactivities of the lysine residues in the glass compared with those previously determined in solution, and therefore, the well-characterized solution reactivity of this reaction cannot be used to predict likely sites of modification in the glassy state. These findings have implications for (i) the selection and combinations of formulation components, particularly with regard to glycation in the glassy state, and (ii) the design of procedures and methodologies for the improvement of protein stability in the glassy state.

Mechanical stability of proteins

A number of experiments and experimentally based simulations showed that beta-proteins are mechanically more stable than alpha-proteins. However, the theory that might explain this evidence is still lacking. In this paper we have developed a simple elastic theory, which allows to estimate critical forces for stretching both kinds of proteins. It has been shown that unfolding of beta-proteins does really require notably higher forces as compared to the stretching of alpha-proteins.