Constant-pH MD simulations of the protonation-triggered conformational switching in diphtheria toxin translocation domain.

Protonation of key residues in the diphtheria toxin translocation (T)-domain triggered by endosomal acidification is critical for inducing a series of conformational transitions critical for the cellular entry of the toxin. Previous experiments revealed the importance of histidine residues in modulating pH-dependent transitions. They suggested the presence of a "safety latch" preventing premature refolding of the T-domain by a yet poorly understood mechanism.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution.

Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein-Lipid Interactions.

Fluorescence spectroscopy provides numerous methodological tools for structural and functional studies of biological macromolecules and their complexes. All fluorescence-based approaches require either existence of an intrinsic probe or an introduction of an extrinsic one. Moreover, studies of complex systems often require an additional introduction of a specific quencher molecule acting in combination with a fluorophore to provide structural or thermodynamic information.

Promoting the activity of a receptor tyrosine phosphatase with a novel pH-responsive transmembrane agonist inhibits cancer-associated phenotypes.

Cell signaling by receptor protein tyrosine kinases (RTKs) is tightly controlled by the counterbalancing actions of receptor protein tyrosine phosphatases (RPTPs). Due to their role in attenuating the signal-initiating potency of RTKs, RPTPs have long been viewed as therapeutic targets. However, the development of activators of RPTPs has remained limited.

Histidine Protonation and Conformational Switching in Diphtheria Toxin Translocation Domain.

Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.

Ukrainian science in the context of its anticolonial struggle.

The current Special Issue entitled "Highlights of Ukrainian Molecular Biosciences" is dedicated to presenting recent contributions in the areas of biochemistry and biophysics, molecular biology and genetics, molecular and cellular physiology, and physical chemistry of biological macromolecules made by researchers either currently working in Ukraine or those who have obtained their training in Ukrainian institutions. Obviously, such a collection can present only a small sample of relevant studies, making the editorial task a particular challenge, as inevitably many deserving research groups were missed. In addition, we are greatly sorrowed that some of the invitees were unable to contribute due to the continued bombardments and military attacks perpetrated by Russia in Ukraine since 2014, and especially in 2022.

Membrane interactions of apoptotic inhibitor Bcl-xL: What can be learned using fluorescence spectroscopy.

Permeabilization of the mitochondrial outer membrane-a point of no return in apoptotic regulation-is tightly controlled by proteins of the Bcl-2 family. Apoptotic inhibitor Bcl-xL is an important member of this family, responsible for blocking the permeabilization, and is also a promising target for anti-cancer drugs. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (i) a soluble folded conformation, (ii) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (iii) refolded membrane-inserted conformations, for which no structural data are available.

Ca and Mg Influence the Thermodynamics of Peptide-Membrane Interactions.

Accurate quantitative estimates of protein-membrane interactions are critical to studies of membrane proteins. Here, we demonstrate that thermodynamic analyses based on current hydropathy scales do not account for the significant and experimentally determined effects that Ca or Mg have on protein-membrane interactions. We examined distinct modes of interaction (interfacial partitioning and folding and transmembrane insertion) by studying three highly divergent peptides: Bid-BH3 (derived from apoptotic regulator Bid), peripherin-2-derived prph2-CTER, and the cancer-targeting pH-Low-Insertion-Peptide (pHLIP).

Ca -dependent interactions between lipids and the tumor-targeting peptide pHLIP.

Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge-based design of cancer-targeting molecular tools, such as pH-low insertion peptide (pHLIP).

Advantages of Quantitative Analysis of Depth-Dependent Fluorescence Quenching: Case Study of BAX.

Dynamic disorder of the lipid bilayer presents a challenge for establishing structure-function relationships in membrane proteins, especially to those that insert by refolding from soluble states, e.g., bacterial toxins and Bcl-2 apoptotic regulators.

Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion.

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown.

Effects of Cardiolipin on the Conformational Dynamics of Membrane-Anchored Bcl-xL.

The anti-apoptotic protein Bcl-xL regulates apoptosis by preventing the permeation of the mitochondrial outer membrane by pro-apoptotic pore-forming proteins, which release apoptotic factors into the cytosol that ultimately lead to cell death. Two different membrane-integrated Bcl-xL constructs have been identified: a membrane-anchored and a membrane-inserted conformation. Here, we use molecular dynamics simulations to study the effect of the mitochondrial specific lipid cardiolipin and the protein protonation state on the conformational dynamics of membrane-anchored Bcl-xL.

Lipids modulate the BH3-independent membrane targeting and activation of BAX and Bcl-xL.

Regulation of apoptosis is tightly linked with the targeting of numerous Bcl-2 proteins to the mitochondrial outer membrane (MOM), where their activation or inhibition dictates cell death or survival. According to the traditional view of apoptotic regulation, BH3-effector proteins are indispensable for the cytosol-to-MOM targeting and activation of proapoptotic and antiapoptotic members of the Bcl-2 protein family. This view is challenged by recent studies showing that these processes can occur in cells lacking BH3 effectors by as yet to be determined mechanism(s).

Conformational switching, refolding and membrane insertion of the diphtheria toxin translocation domain.

Diphtheria toxin is among many bacterial toxins that utilize the endosomal pathway of cellular entry, which is ensured by the bridging of the endosomal membrane by the toxin's translocation (T) domain. Endosomal acidification triggers a series of conformational changes of the T-domain, that take place first in aqueous and subsequently in membranous milieu. These rearrangements ultimately result in establishing membrane-inserted conformation(s) and translocation of the catalytic moiety of the toxin into the cytoplasm.

Expanding MPEx Hydropathy Analysis to Account for Electrostatic Contributions to Protein Interactions with Anionic Membranes.

Hydropathy plots are a crucial tool to guide experimental design, as they generate predictions of protein-membrane interactions and their bilayer topology. The predictions are based on experimentally determined hydrophobicity scales, which provide an estimate for the propensity and stability of these interactions. A significant improvement to the accuracy of hydropathy analyses was provided by the development of the popular Wimley-White interfacial and octanol hydrophobicity scales.

Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain.

Diphtheria toxin, an exotoxin secreted by that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition.

Regulation of Apoptosis by the Bcl-2 Family of Proteins: Field on a Brink.

Apoptosis, a form of programmed cell death, is a highly regulated process critical for tissue development, homeostasis, and pathogenesis of various diseases [...

Correction to: Experimental and Computational Characterization of Oxidized and Reduced Protegrin Pores in Lipid Bilayers.

The original version of the article was published without the Graphic Abstract. Graphic Abstract image of the article is given below.

Experimental and Computational Characterization of Oxidized and Reduced Protegrin Pores in Lipid Bilayers.

Protegrin-1 (PG-1), an 18-residue β-hairpin stabilized by two disulfide bonds, is a member of a family of powerful antimicrobial peptides which are believed to act through membrane permeabilization. Here we used a combination of experimental and computational approaches to characterize possible structural arrangements of PG-1 in lipid bilayers mimicking bacterial membranes. We have measured the dose-response function of the PG-1-induced leakage of markers of various sizes from vesicles and found it to be consistent with the formation of pores of two different sizes.

Conformational Switching in Bcl-xL: Enabling Non-Canonic Inhibition of Apoptosis Involves Multiple Intermediates and Lipid Interactions.

The inhibition of mitochondrial permeabilization by the anti-apoptotic protein Bcl-xL is crucial for cell survival and homeostasis. Its inhibitory role requires the partitioning of Bcl-xL to the mitochondrial outer membrane from an inactive state in the cytosol, leading to its extensive refolding. The molecular mechanisms behind these events and the resulting conformations in the bilayer are unclear, and different models have been proposed to explain them.

Divalent Cations and Lipid Composition Modulate Membrane Insertion and Cancer-Targeting Action of pHLIP.

The pH-Low Insertion Peptide (pHLIP) has emerged as an important tool for targeting cancer cells; it has been assumed that its targeting mechanism depends solely on the mild acidic environment surrounding tumors. Here, we examine the role of Ca and Mg on pHLIP's insertion, cellular targeting, and drug delivery. We demonstrate that physiologically relevant concentrations of either cation can shift the protonation-dependent transition by up to several pH units toward basic pH and induce substantial protonation-independent transmembrane insertion of pHLIP at pH as high as 10.

Location of TEMPO-PC in Lipid Bilayers: Implications for Fluorescence Quenching.

The characterization of the behavior of lipid-attached spin probes in a bilayer is of fundamental importance for correct interpretation of the results of both EPR and fluorescence studies of protein-membrane interactions. The knowledge of the immersion depth of TEMPO spin probe attached to lipid headgroup in TEMPO-PC is critical for the determination of the transverse location of fluorescence probes attached to proteins and peptides. The question of bilayer penetration of TEMPO moiety in TEMPO-PC has recently came into prominence in two studies of interfacial solvation (Cheng et al.