We record proton NMR spectra of the protein ubiquitin at 1 atmosphere pressure and at negative pressures (under tension), under conditions where the native and denatured states are nearly equally populated. Analysis of the unique histidine aromatic resonance of ubiquitin shows that negative pressure destabilizes the protein, in accord with a quadratic free energy dependence on pressure and temperature previously suggested in the literature. Our molecular dynamics simulations at negative pressure agree with the experimental result. In addition, molecular dynamics predicts a turnaround of the folding free energy at very low pressure. An 'island of stability' may exist at very negative pressures, where the protein is likely to fold into low density fluctuations of the solvent.

Protein stability assessment of seven model proteins in the presence of low molecular weight polyethyleneimine (PEI, MW 2000 Da) was performed. Thermodynamic stability, monitored by circular dichroism (CD) spectroscopy, showed that the polymer did not have a major effect on the melting temperature (T(m)) of the basic proteins - muscle lactate dehydrogenase (LDH), ribonuclease A, lysozyme and cutinase, while for the acidic ones - human growth hormone, human serum albumin and heart lactate dehydrogenase - there was a shift in T(m) towards lower temperatures. The secondary structures of the basic proteins were essentially the same, with none or a slight increase in the CD spectra, in presence of the polymer. In the case of the acidic proteins, the CD spectra were diminished mostly due to phase separation. Assuming a homogeneous distribution of the net charge on the protein surface a quantitative inverse relationship was established between surface charge density of the acidic proteins and the PEI(2000) concentration required for maximum flocculation. Despite lowering the thermal stability of acidic proteins, PEI(2000) was seen to protect heart LDH at an increasing oxidative stress.

Proteins have evolved to balance efficient binding of desired partners with rejection of unwanted interactions. To investigate the evolution of protein-protein interactions, we selected a random library of pre-stabilized TEM1 β-lactamase against wild-type TEM1 using yeast surface display. Three mutations were sufficient to achieve micromolar affinity binding between the two. The X-ray structure emphasized that the main contribution of the selected mutations was to modify the protein fold, specifically removing the N'-terminal helix, which consequently allowed protein coupling via a β-sheet-mediated interaction resembling amyloid interaction mode. The only selected mutation located at the interaction interface (E58V) is reminiscent of the single mutation commonly causing sickle-cell anemia. Interestingly, the evolved mutations cannot be inserted into the wild-type protein due to reduced thermal stability of the resulting mutant protein. These results reveal a simple mechanism by which undesirable binding is purged by loss of thermal stability.

Short half-life proteins regulate many essential processes, including cell cycle, transcription, and apoptosis. However, few well-characterized protein-turnover pathways have been identified because traditional methods to measure protein half-life are time and labor intensive. To overcome this barrier, we developed a protein stability probe and high-content screening pipeline for novel regulators of short half-life proteins using automated image analysis. Our pilot probe consists of the short half-life protein c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus). This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. Rapid turnover prevents maximal fluorescence of the probe due to the relatively longer maturation time of the fluorescent protein. Cells expressing the MYC-Venus probe were analyzed using a pipeline in which automated confocal microscopy and image analyses were used to score MYC-Venus stability by two strategies: assaying the percentage of cells with Venus fluorescence above background, and phenotypic comparative analysis. To evaluate this high-content screening pipeline and our probe, a kinase inhibitor library was screened by confocal microscopy to identify known and novel kinases that regulate MYC stability. Compounds identified were shown to increase the half-life of both MYC-Venus and endogenous MYC, validating the probe and pipeline. Fusion of another short half-life protein, myeloid cell leukemia 1 (MCL1), with Venus also demonstrated an increase in percent Venus-positive cells after treatment with inhibitors known to stabilize MCL1. Together, the results validate the use of our automated microscopy and image analysis pipeline of stability probe-expressing cells to rapidly and quantitatively identify regulators of short half-life proteins. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Stability is one of the most fundamental intrinsic characteristics of proteins and can be determined with various methods. Characterization of protein properties does not keep pace with increase in new sequence data and therefore even basic properties are not known for far majority of identified proteins. There have been some attempts to develop predictors for protein stabilities; however, they have suffered from small numbers of known examples.

Posttranslational modifications offer a dynamic way to regulate protein activity, subcellular localization, and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites tend to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. Analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and that binding hotspots tend to be phosphorylated in heterooligomers. Although the majority of complexes do not show significant estimated stability differences upon phosphorylation or dephosphorylation, for about one-third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are more likely to be evolutionary conserved than other interfacial residues.

The anti-apoptotic myeloid cell leukemia 1 (MCL1) protein belongs to the pro-survival BCL2 family and is frequently amplified or elevated in human cancers. MCL1 is highly unstable, with its stability being regulated by phosphorylation and ubiquitination. Here, we identify acetylation as another critical post-translational modification regulating MCL1 protein stability. We demonstrate that the lysine acetyltransferase p300 targets MCL1 at K40 for acetylation, which is counteracted by the deacetylase sirtuin 3 (SIRT3). Mechanistically, acetylation enhances MCL1 interaction with USP9X, resulting in deubiquitination and subsequent MCL1 stabilization. Therefore, ectopic expression of acetylation-mimetic MCL1 promotes apoptosis evasion of cancer cells, enhances colony formation potential, and facilitates xenografted tumor progression. We further demonstrate that elevated MCL1 acetylation sensitizes multiple cancer cells to pharmacological inhibition of USP9X. These findings reveal that acetylation of MCL1 is a critical post-translational modification enhancing its oncogenic function and provide a rationale for developing innovative therapeutic strategies for MCL1-dependent tumors.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an obligate pathogen that causes 10.4 million new infections worldwide, out of which about 1.4 million die every year. SDS is routinely used to mimic the native hydrophobic environment of phospholipid bilayer. Here, we report structure and stability of a mammalian cell entry protein from M. tuberculosis (Mce4A) in the absence and presence of SDS. The far-UV circular dichroism (CD) measurements suggested that SDS induces α-helical structure in Mce4A. Stability of the protein in the absence and presence of SDS was measured from the analysis of the urea-induced denaturation curves of three physical properties (CD, intrinsic fluorescence and near-UV absorption). These measurements led to the conclusion that SDS stabilizes Mce4A. Binding of SDS with Mce4A was measured in isothermal titration calorimeter, which led to the conclusion that there is strong binding of SDS with Mce4A. We propose that the membrane associated Mce4A is more structured and more stable.

Protein scientists have long used cosolutes to study protein stability. While denaturants, such as urea, have been employed for a long time, the attention became focused more recently on protein stabilizers, including osmolytes. Here, we provide practical experimental instructions for the use of both stabilizing and denaturing osmolytes with proteins, as well as data evaluation strategies. We focus on protein stability in the presence of cosolutes and their mixtures at constant and variable temperature.

REDD1 is known to be transcriptionally upregulated in hypoxia. During hypoxic stress, REDD1 plays an important role as a mediator of mTORC1 inhibition. REDD1 is also subject to highly dynamic transcriptional regulation in response to a variety of other stress signals. In addition, the REDD1 protein is highly unstable. However, it is currently not well understood how REDD1 protein stability is regulated. In this study, we discovered that mTORC1 regulates REDD1 protein stability in a 26S proteasome dependent manner. Inhibition of mTORC1 resulted in reduced REDD1 protein stability and a consequent decrease in REDD1 expression. Conversely, activation of the mTORC1 pathway increases REDD1 protein levels. We show that REDD1 degradation is not regulated by HUWE1, Cul4a or other Cullin E3 ubiquitin ligases. Our study shows that mTORC1 increases REDD1 protein stability and reveals a novel mTORC1-REDD1 feedback loop. This feedback mechanism may limit the inhibitory action of REDD1 on mTORC1.

Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of [Formula: see text]-helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/ .

Although chloroplast protein stability has long been recognised as a major level of post-translational regulation in photosynthesis and gene expression, the factors determining protein stability in plastids are largely unknown. Here, we have identified stability determinants in vivo by producing plants with transgenic chloroplasts that express a reporter protein whose N- and C-termini were systematically modified. We found that major stability determinants are located in the N-terminus. Moreover, testing of all 20 amino acids in the position after the initiator methionine revealed strong differences in protein stability and indicated an important role of the penultimate N-terminal amino acid residue in determining the protein half life. We propose that the stability of plastid proteins is largely determined by three factors: (i) the action of methionine aminopeptidase (the enzyme that removes the initiator methionine and exposes the penultimate N-terminal amino acid residue), (ii) an N-end rule-like protein degradation pathway, and (iii) additional sequence determinants in the N-terminal region.

Understanding the structural basis of protein-protein interactions (PPIs) may shed light on the organization and functioning of signal transduction and metabolic networks and may assist in structure-based design of ligands (drugs) targeting protein-protein interfaces. The residues at the bimolecular interface, designated as the hot spots, contribute most of the free binding energy of PPI. To date, there is no conclusive atomistic explanation for the unique functional properties of the hot spots. We hypothesized that backbone compliance may play a role in protein-protein recognition and in the mechanism of binding of small-molecule compounds to protein surfaces. We used a steered molecular dynamics simulation to explore the compliance properties of the backbone of surface-exposed residues in several model proteins: interleukin-2, mouse double minute protein 2 and proliferating cell nuclear antigen. We demonstrated that protein surfaces exhibit distinct patterns in which highly immobile residues form defined clusters ("stability patches") alternating with areas of moderate to high mobility. These "stability patches" tend to localize in functionally important regions involved in protein-protein recognition. We propose a mechanism by which the distinct structural organization of the hot spots may contribute to their role in mediating PPI and facilitating binding of structurally diverse small-molecule compounds to protein surfaces.

Predicting the thermodynamic stability of proteins is a common and widely used step in protein engineering, and when elucidating the molecular mechanisms behind evolution and disease. Here, we present RaSP, a method for making rapid and accurate predictions of changes in protein stability by leveraging deep learning representations. RaSP performs on-par with biophysics-based methods and enables saturation mutagenesis stability predictions in less than a second per residue. We use RaSP to calculate ∼ 230 million stability changes for nearly all single amino acid changes in the human proteome, and examine variants observed in the human population. We find that variants that are common in the population are substantially depleted for severe destabilization, and that there are substantial differences between benign and pathogenic variants, highlighting the role of protein stability in genetic diseases. RaSP is freely available-including via a Web interface-and enables large-scale analyses of stability in experimental and predicted protein structures.

Every amino acid residue can influence a protein's overall stability, making stability highly susceptible to change throughout evolution. We consider the distribution of protein stabilities evolutionarily permittable under two previously reported protein fitness functions: flux dynamics and misfolding avoidance. We develop an evolutionary dynamics theory and find that it agrees better with an extensive protein stability data set for dihydrofolate reductase orthologs under the misfolding avoidance fitness function rather than the flux dynamics fitness function. Further investigation with ribonuclease H data demonstrates that not any misfolded state is avoided; rather, it is only the unfolded state. At the end, we discuss how our work pertains to the universal protein abundance-evolutionary rate correlation seen across organisms' proteomes. We derive a closed-form expression relating protein abundance to evolutionary rate that captures Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens experimental trends without fitted parameters.

Genetic recombination is a common evolutionary mechanism that produces molecular diversity. However, its consequences on protein folding stability have not attracted the same attention as in the case of point mutations. Here, we studied the effects of homologous recombination on the computationally predicted protein folding stability for several protein families, finding less detrimental effects than we previously expected. Although recombination can affect multiple protein sites, we found that the fraction of recombined proteins that are eliminated by negative selection because of insufficient stability is not significantly larger than the corresponding fraction of proteins produced by mutation events. Indeed, although recombination disrupts epistatic interactions, the mean stability of recombinant proteins is not lower than that of their parents. On the other hand, the difference of stability between recombined proteins is amplified with respect to the parents, promoting phenotypic diversity. As a result, at least one third of recombined proteins present stability between those of their parents, and a substantial fraction have higher or lower stability than those of both parents. As expected, we found that parents with similar sequences tend to produce recombined proteins with stability close to that of the parents. Finally, the simulation of protein evolution along the ancestral recombination graph with empirical substitution models commonly used in phylogenetics, which ignore constraints on protein folding stability, showed that recombination favors the decrease of folding stability, supporting the convenience of adopting structurally constrained models when possible for inferences of protein evolutionary histories with recombination.

Glycine (Gly) residues are particularly susceptible to hydrogen abstraction; which results in the formation of the capto-dative stabilized Cα-centered Gly radical (GLR) on the protein backbone. We examined the effect of GLR formation on the structure of the Trp cage; tryptophan zipper; and the villin headpiece; three fast-folding and stable miniproteins; using all-atom (OPLS-AA) molecular dynamics simulations. Radicalization changes the conformation of the GLR residue and affects both neighboring residues but did not affect the stability of the Trp zipper. The stability of helices away from the radical center in villin were also affected by radicalization; and GLR in place of Gly15 caused the Trp cage to unfold within 1 µs. These results provide new evidence on the destabilizing effects of protein oxidation by reactive oxygen species.

BRCA1 is frequently down-regulated in breast cancer, the underlying mechanism is unclear. Here we identified DCAF8L1, an X-linked gene product, as a DDB1-Cullin associated Factor (DCAF) for CUL4 E3 ligases to target BRCA1 and BARD1 for proteasomal degradation. Forced expression of DCAF8L1 caused reduction of BRCA1 and BARD1, and impaired DNA damage repair function, conferring increased sensitivity to irradiation and DNA damaging agents, as well as Olaparib, a PARPi anticancer drug; while depletion of DCAF8L1 restored BRCA1 and suppressed the growth of its xenograft tumors. Furthermore, the expression of DCAF8L1 was induced in human H9 ES cells during transition from primed to naïve state when Xi chromosome was reactivated. Aberrant expression of DCAF8L1 was observed in human breast fibroadenoma and breast cancer. These findings suggest that CRL4DCAF8L1 is an important E3 ligase that may participate in the development of breast cancer, probably through regulating the stability of BRCA1 and BARD1 tumor suppressor, linking BRCA1 and X chromosome inactivation to breast carcinogenesis.

Halophile proteins can tolerate high salt concentrations. Understanding halophilicity features is the first step toward engineering halostable crops. To this end, we examined protein features contributing to the halo-toleration of halophilic organisms. We compared more than 850 features for halophilic and non-halophilic proteins with various screening, clustering, decision tree, and generalized rule induction models to search for patterns that code for halo-toleration. Up to 251 protein attributes selected by various attribute weighting algorithms as important features contribute to halo-stability; from them 14 attributes selected by 90% of models and the count of hydrogen gained the highest value (1.0) in 70% of attribute weighting models, showing the importance of this attribute in feature selection modeling. The other attributes mostly were the frequencies of di-peptides. No changes were found in the numbers of groups when K-Means and TwoStep clustering modeling were performed on datasets with or without feature selection filtering. Although the depths of induced trees were not high, the accuracies of trees were higher than 94% and the frequency of hydrophobic residues pointed as the most important feature to build trees. The performance evaluation of decision tree models had the same values and the best correctness percentage recorded with the Exhaustive CHAID and CHAID models. We did not find any significant difference in the percent of correctness, performance evaluation, and mean correctness of various decision tree models with or without feature selection. For the first time, we analyzed the performance of different screening, clustering, and decision tree algorithms for discriminating halophilic and non-halophilic proteins and the results showed that amino acid composition can be used to discriminate between halo-tolerant and halo-sensitive proteins.

Protein methylation is an important modification beyond epigenetics. However, systems analyses of protein methylation lag behind compared to other modifications. Recently, thermal stability analyses have been developed which provide a proxy of a protein functional status. Here, we show that molecular and functional events closely linked to protein methylation can be revealed by the analysis of thermal stability. Using mouse embryonic stem cells as a model, we show that Prmt5 regulates mRNA binding proteins that are enriched in intrinsically disordered regions and involved in liquid-liquid phase separation mechanisms, including the formation of stress granules. Moreover, we reveal a non-canonical function of Ezh2 in mitotic chromosomes and the perichromosomal layer, and identify Mki67 as a putative Ezh2 substrate. Our approach provides an opportunity to systematically explore protein methylation function and represents a rich resource for understanding its role in pluripotency.