Quantitative entropy-enthalpy compensation in intraprotein interactions from model compound data.

Many small globular proteins exist in only two states-the physiologically relevant folded state and an inactive unfolded state. The active state is stabilized by numerous weak attractive contacts, including hydrogen bonds, other polar interactions, and the hydrophobic effect. Knowledge of these interactions is key to understanding the fundamental equilibrium thermodynamics of protein folding and stability. We focus on one such interaction, that between amide and aromatic groups. We provide a statistically convincing case for quantitative, linear entropy-enthalpy compensation in forming aromatic-amide interactions using published model compound transfer-free energy data.

On the role of native contact cooperativity in protein folding.

The consistency of energy landscape theory predictions with available experimental data, as well as direct evidence from molecular simulations, have shown that protein folding mechanisms are largely determined by the contacts present in the native structure. As expected, native contacts are generally energetically favorable. However, there are usually at least as many energetically favorable nonnative pairs owing to the greater number of possible nonnative interactions. This apparent frustration must therefore be reduced by the greater cooperativity of native interactions. In this work, we analyze the statistics of contacts in the unbiased all-atom folding trajectories obtained by Shaw and coworkers, focusing on the unfolded state. By computing mutual cooperativities between contacts formed in the unfolded state, we show that native contacts form the most cooperative pairs, while cooperativities among nonnative or between native and nonnative contacts are typically much less favorable or even anticooperative. Furthermore, we show that the largest network of cooperative interactions observed in the unfolded state consists mainly of native contacts, suggesting that this set of mutually reinforcing interactions has evolved to stabilize the native state.

The S-component fold: a link between bacterial transporters and receptors.

The processes of nutrient uptake and signal sensing are crucial for microbial survival and adaptation. Membrane-embedded proteins involved in these functions (transporters and receptors) are commonly regarded as unrelated in terms of sequence, structure, mechanism of action and evolutionary history. Here, we analyze the protein structural universe using recently developed artificial intelligence-based structure prediction tools, and find an unexpected link between prominent groups of microbial transporters and receptors. The so-called S-components of Energy-Coupling Factor (ECF) transporters, and the membrane domains of sensor histidine kinases of the 5TMR cluster share a structural fold. The discovery of their relatedness manifests a widespread case of prokaryotic "transceptors" (related proteins with transport or receptor function), showcases how artificial intelligence-based structure predictions reveal unchartered evolutionary connections between proteins, and provides new avenues for engineering transport and signaling functions in bacteria.

Calcium-induced structural transitions are central to the folding, function, and processing of serratiopeptidase zymogen into mature form.

Serratia marcescens is an emerging health-threatening, gram-negative opportunistic pathogen associated with a wide variety of localized and life-threatening systemic infections. One of the most crucial virulence factors produced by S. marcescens is serratiopeptidase, a 50.2-kDa repeats-in-toxin (RTX) family broad-specificity zinc metalloprotease. RTX family proteins are functionally diverse exoproteins of gram-negative bacteria that exhibit calcium-dependent structural dynamicity and are secreted through a common type-1 secretion system (T1SS) machinery. To evaluate the impact of various divalent ligands on the folding and maturation of serratiopeptidase zymogen, the protein was purified and a series of structural and functional investigations were undertaken. The results indicate that calcium binding to the C-terminal RTX domain acts as a folding switch, triggering a disordered-to-ordered transition in the enzyme's conformation. Further, the auto-processing of the 16-amino acid N-terminal pro-peptide results in the maturation of the enzyme. The binding of calcium ions to serratiopeptidase causes a highly cooperative conformational transition in its structure, which is essential for the enzyme's activation and maturation. This conformational change is accompanied by an increase in solubility and enzymatic activity. For efficient secretion and to minimize intracellular toxicity, the enzyme needs to be in an unfolded extended form. The calcium-rich extracellular environment favors the folding and processing of zymogen into mature serratiopeptidase, i.e., the holo-form required by S. marcescens to establish infections and survive in different environmental niches.

LoCoHD: a metric for comparing local environments of proteins.

Protein folds and the local environments they create can be compared using a variety of differently designed measures, such as the root mean squared deviation, the global distance test, the template modeling score or the local distance difference test. Although these measures have proven to be useful for a variety of tasks, each fails to fully incorporate the valuable chemical information inherent to atoms and residues, and considers these only partially and indirectly. Here, we develop the highly flexible local composition Hellinger distance (LoCoHD) metric, which is based on the chemical composition of local residue environments. Using LoCoHD, we analyze the chemical heterogeneity of amino acid environments and identify valines having the most conserved-, and arginines having the most variable chemical environments. We use LoCoHD to investigate structural ensembles, to evaluate critical assessment of structure prediction (CASP) competitors, to compare the results with the local distance difference test (lDDT) scoring system, and to evaluate a molecular dynamics simulation. We show that LoCoHD measurements provide unique information about protein structures that is distinct from, for example, those derived using the alignment-based RMSD metric, or the similarly distance matrix-based but alignment-free lDDT metric.

Oxidative photocatalysis on membranes triggers non-canonical pyroptosis.

Intracellular membranes composing organelles of eukaryotes include membrane proteins playing crucial roles in physiological functions. However, a comprehensive understanding of the cellular responses triggered by intracellular membrane-focused oxidative stress remains elusive. Herein, we report an amphiphilic photocatalyst localised in intracellular membranes to damage membrane proteins oxidatively, resulting in non-canonical pyroptosis. Our developed photocatalysis generates hydroxyl radicals and hydrogen peroxides via water oxidation, which is accelerated under hypoxia. Single-molecule magnetic tweezers reveal that photocatalysis-induced oxidation markedly destabilised membrane protein folding. In cell environment, label-free quantification reveals that oxidative damage occurs primarily in membrane proteins related to protein quality control, thereby aggravating mitochondrial and endoplasmic reticulum stress and inducing lytic cell death. Notably, the photocatalysis activates non-canonical inflammasome caspases, resulting in gasdermin D cleavage to its pore-forming fragment and subsequent pyroptosis. These findings suggest that the oxidation of intracellular membrane proteins triggers non-canonical pyroptosis.

Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

Getting membrane proteins into shape.

In this issue, Ji et al.1 show how a multipass membrane protein that initially inserts into the endoplasmic reticulum in a mostly inverted topology is post-translationally dislocated, re-inserted, and folded with the help of ATP13A1, a P-type ATPase.

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

A finely balanced order-disorder equilibrium sculpts the folding-binding landscape of an antibiotic sequestering protein.

TipA, a MerR family transcription factor from Streptomyces lividans, promotes antibiotic resistance by sequestering broad-spectrum thiopeptide-based antibiotics, thus counteracting their inhibitory effect on ribosomes. TipAS, a minimal binding motif which is expressed as an isoform of TipA, harbors a partially disordered N-terminal subdomain that folds upon binding multiple antibiotics. The extent and nature of the underlying molecular heterogeneity in TipAS that shapes its promiscuous folding-function landscape is an open question and is critical for understanding antibiotic-sequestration mechanisms. Here, combining equilibrium and time-resolved experiments, statistical modeling, and simulations, we show that the TipAS native ensemble exhibits a pre-equilibrium between binding-incompetent and binding-competent substates, with the fully folded state appearing only as an excited state under physiological conditions. The binding-competent state characterized by a partially structured N-terminal subdomain loses structure progressively in the physiological range of temperatures, swells on temperature increase, and displays slow conformational exchange across multiple conformations. Binding to the bactericidal antibiotic thiostrepton follows a combination of induced-fit and conformational-selection-like mechanisms, via partial binding and concomitant stabilization of the binding-competent substate. These ensemble features are evolutionarily conserved across orthologs from select bacteria that infect humans, underscoring the functional role of partial disorder in the native ensemble of antibiotic-sequestering proteins belonging to the MerR family.

Electrostatics introduce a trade-off between mesophilic stability and adaptation in halophilic proteins.

Extremophile organisms have adapted to extreme physicochemical conditions. Halophilic organisms, in particular, survive at very high salt concentrations. To achieve this, they have engineered the surface of their proteins to increase the number of short, polar and acidic amino acids, while decreasing large, hydrophobic and basic residues. While these adaptations initially decrease protein stability in the absence of salt, they grant halophilic proteins remarkable stability in environments with extremely high salt concentrations, where non-adapted proteins unfold and aggregate. The molecular mechanisms by which halophilic proteins achieve this, however, are not yet clear. Here, we test the hypothesis that the halophilic amino acid composition destabilizes the surface of the protein, but in exchange improves the stability in the presence of salts. To do that, we have measured the folding thermodynamics of various protein variants with different degrees of halophilicity in the absence and presence of different salts, and at different pH values to tune the ionization state of the acidic amino acids. Our results show that halophilic amino acids decrease the stability of halophilic proteins under mesophilic conditions, but in exchange improve salt-induced stabilization and solubility. We also find that, in contrast to traditional assumptions, contributions arising from hydrophobic effect and preferential ion exclusion are more relevant for haloadaptation than electrostatics. Overall, our findings suggest a trade-off between folding thermodynamics and halophilic adaptation to optimize proteins for hypersaline environments.

HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.

Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes.

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.

Unraveling the Connection: Pain and Endoplasmic Reticulum Stress.

Pain is a complex and multifaceted experience. Recent research has increasingly focused on the role of endoplasmic reticulum (ER) stress in the induction and modulation of pain. The ER is an essential organelle for cells and plays a key role in protein folding and calcium dynamics. Various pathological conditions, such as ischemia, hypoxia, toxic substances, and increased protein production, may disturb protein folding, causing an increase in misfolding proteins in the ER. Such an overload of the folding process leads to ER stress and causes the unfolded protein response (UPR), which increases folding capacity in the ER. Uncompensated ER stress impairs intracellular signaling and cell function, resulting in various diseases, such as diabetes and degenerative neurological diseases. ER stress may be a critical universal mechanism underlying human diseases. Pain sensations involve the central as well as peripheral nervous systems. Several preclinical studies indicate that ER stress in the nervous system is enhanced in various painful states, especially in neuropathic pain conditions. The purpose of this narrative review is to uncover the intricate relationship between ER stress and pain, exploring molecular pathways, implications for various pain conditions, and potential therapeutic strategies.

Diversifying de novo TIM barrels by hallucination.

De novo protein design expands the protein universe by creating new sequences to accomplish tailor-made enzymes in the future. A promising topology to implement diverse enzyme functions is the ubiquitous TIM-barrel fold. Since the initial de novo design of an idealized four-fold symmetric TIM barrel, the family of de novo TIM barrels is expanding rapidly. Despite this and in contrast to natural TIM barrels, these novel proteins lack cavities and structural elements essential for the incorporation of binding sites or enzymatic functions. In this work, we diversified a de novo TIM barrel by extending multiple ßα-loops using constrained hallucination. Experimentally tested designs were found to be soluble upon expression in Escherichia coli and well-behaved. Biochemical characterization and crystal structures revealed successful extensions with defined α-helical structures. These diversified de novo TIM barrels provide a framework to explore a broad spectrum of functions based on the potential of natural TIM barrels.

Folding Kinetics and Volume Variation of the ß-Hairpin Peptide Chignolin upon Ultrafast pH-Jumps.

In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex biomacromolecules. We have previously reported on the folding/unfolding kinetics of a model α-helix. Here, we study folding transitions in chignolin (GYDPETGTWG), a short ß-hairpin peptide previously used as a model to study conformational changes in ß-sheet proteins. Although previously suggested, until now, the role of the Tyr2-Trp9 interaction in the folding mechanism of chignolin was not clear. In the present work, pH-dependent conformational changes of chignolin were characterized by circular dichroism (CD), nuclear magnetic resonance (NMR), ultrafast pH-jump coupled with time-resolved photoacoustic calorimetry (TR-PAC), and molecular dynamics (MD) simulations. Taken together, our results present a comprehensive view of chignolin's folding kinetics upon local pH changes and the role of the Tyr2-Trp9 interaction in the folding process. CD data show that chignolin's ß-hairpin formation displays a pH-dependent skew bell-shaped curve, with a maximum close to pH 6, and a large decrease in ß-sheet content at alkaline pH. The ß-hairpin structure is mainly stabilized by aromatic interactions between Tyr2 and Trp9 and CH-π interactions between Tyr2 and Pro4. Unfolding of chignolin at high pH demonstrates that protonation of Tyr2 is essential for the stability of the ß-hairpin. Refolding studies were triggered by laser-induced pH-jumps and detected by TR-PAC. The refolding of chignolin from high pH, mainly due to the protonation of Tyr2, is characterized by a volume expansion (10.4 mL mol-1), independent of peptide concentration, in the microsecond time range (lifetime of 1.15 µs). At high pH, the presence of the deprotonated hydroxyl (tyrosinate) hinders the formation of the aromatic interaction between Tyr2 and Trp9 resulting in a more disorganized and dynamic tridimensional structure of the peptide. This was also confirmed by comparing MD simulations of chignolin under conditions mimicking neutral and high pH.

Innovative peptide architectures: advancements in foldamers and stapled peptides for drug discovery.

INTRODUCTION: Peptide foldamers play a critical role in pharmaceutical research and biomedical applications. This review highlights recent (post-2020) advancements in novel foldamers, synthetic techniques, and their applications in pharmaceutical research. AREAS COVERED: The authors summarize the structures and applications of peptide foldamers such as α, ß, γ-peptides, hydrocarbon-stapled peptides, urea-type foldamers, sulfonic-γ-amino acid foldamers, aromatic foldamers, and peptoids, which tackle the challenges of traditional peptide drugs. Regarding antimicrobial use, foldamers have shown progress in their potential against drug-resistant bacteria. In drug development, peptide foldamers have been used as drug delivery systems (DDS) and protein-protein interaction (PPI) inhibitors. EXPERT OPINION: These structures exhibit resistance to enzymatic degradation, are promising for therapeutic delivery, and disrupt crucial PPIs associated with diseases such as cancer with specificity, versatility, and stability, which are useful therapeutic properties. However, the complexity and cost of their synthesis, along with the necessity for thorough safety and efficacy assessments, necessitate extensive research and cross-sector collaboration. Advances in synthesis methods, computational modeling, and targeted delivery systems are essential for fully realizing the therapeutic potential of foldamers and integrating them into mainstream medical treatments.

Studying protein stability in crowded environments by NMR.

Most proteins perform their functions in crowded and complex cellular environments where weak interactions are ubiquitous between biomolecules. These complex environments can modulate the protein folding energy landscape and hence affect protein stability. NMR is a nondestructive and effective method to quantify the kinetics and equilibrium thermodynamic stability of proteins at an atomic level within crowded environments and living cells. Here, we review NMR methods that can be used to measure protein stability, as well as findings of studies on protein stability in crowded environments mimicked by polymer and protein crowders and in living cells. The important effects of chemical interactions on protein stability are highlighted and compared to spatial excluded volume effects.

The evolutionary novelty of insect defensins: from bacterial killing to toxin neutralization.

Insect host defense comprises two complementary dimensions, microbial killing-mediated resistance and microbial toxin neutralization-mediated resilience, both jointly providing protection against pathogen infections. Insect defensins are a class of effectors of innate immunity primarily responsible for resistance to Gram-positive bacteria. Here, we report a newly originated gene from an ancestral defensin via genetic deletion following gene duplication in Drosophila virilis, which confers an enhanced resilience to Gram-positive bacterial infection. This gene encodes an 18-mer arginine-rich peptide (termed DvirARP) with differences from its parent gene in its pattern of expression, structure and function. DvirARP specifically expresses in D. virilis female adults with a constitutive manner. It adopts a novel fold with a 310 helix and a two CXC motif-containing loop stabilized by two disulfide bridges. DvirARP exhibits no activity on the majority of microorganisms tested and only a weak activity against two Gram-positive bacteria. DvirARP knockout flies are viable and have no obvious defect in reproductivity but they are more susceptible to the DvirARP-resistant Staphylococcus aureus infection than the wild type files, which can be attributable to its ability in neutralization of the S. aureus secreted toxins. Phylogenetic distribution analysis reveals that DvirARP is restrictedly present in the Drosophila subgenus, but independent deletion variations also occur in defensins from the Sophophora subgenus, in support of the evolvability of this class of immune effectors. Our work illustrates for the first time how a duplicate resistance-mediated gene evolves an ability to increase the resilience of a subset of Drosophila species against bacterial infection.