Analysis of protein-protein and protein-membrane interactions by isotope-edited infrared spectroscopy.

The objective of this work is to highlight the power of isotope-edited Fourier transform infrared (FTIR) spectroscopy in resolving important problems encountered in biochemistry, biophysics, and biomedical research, focusing on protein-protein and protein membrane interactions that play key roles in practically all life processes. An overview of the effects of isotope substitutions in (bio)molecules on spectral frequencies and intensities is given. Data are presented demonstrating how isotope-labeled proteins and/or lipids can be used to elucidate enzymatic mechanisms, the mode of membrane binding of peripheral proteins, regulation of membrane protein function, protein aggregation, and local and global structural changes in proteins during functional transitions.

The structure of tyrosine-10 favors ionic conductance of Alzheimer's disease-associated full-length amyloid-β channels.

Amyloid β (Aβ) ion channels destabilize cellular ionic homeostasis, which contributes to neurotoxicity in Alzheimer's disease. The relative roles of various Aβ isoforms are poorly understood. We use bilayer electrophysiology, AFM imaging, circular dichroism, FTIR and fluorescence spectroscopy to characterize channel activities of four most prevalent Aβ peptides, Aβ, Aβ, and their pyroglutamylated forms (AβpE, AβpE) and correlate them with the peptides' structural features.

Transcriptional mutagenesis of α-synuclein caused by DNA oxidation in Parkinson's disease pathogenesis.

Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown.

Acid-induced disassembly of the Haemophilus ducreyi cytolethal distending toxin.

The cytolethal distending toxins (CDTs) produced by many Gram-negative pathogens are tripartite genotoxins with a single catalytic subunit (CdtB) and two cell-binding subunits (CdtA + CdtC). CDT moves by vesicle carriers from the cell surface to the endosomes and through the Golgi apparatus en route to the endoplasmic reticulum (ER). CdtA dissociates from the rest of the toxin before reaching the Golgi apparatus, and CdtB separates from CdtC in the ER.

Challenges and hopes for Alzheimer's disease.

Recent drug development efforts targeting Alzheimer's disease (AD) have failed to produce effective disease-modifying agents for many reasons, including the substantial presymptomatic neuronal damage that is caused by the accumulation of the amyloid β (Aβ) peptide and tau protein abnormalities, deleterious adverse effects of drug candidates, and inadequate design of clinical trials. New molecular targets, biomarkers, and diagnostic techniques, as well as alternative nonpharmacological approaches, are sorely needed to detect and treat early pathological events. This article analyzes the successes and debacles of pharmaceutical endeavors to date, and highlights new technologies that may lead to the more effective diagnosis and treatment of the pathologies that underlie AD.

Holotoxin disassembly by protein disulfide isomerase is less efficient for Escherichia coli heat-labile enterotoxin than cholera toxin.

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally similar AB-type protein toxins. They move from the cell surface to the endoplasmic reticulum where the A1 catalytic subunit is separated from its holotoxin by protein disulfide isomerase (PDI), thus allowing the dissociated A1 subunit to enter the cytosol for a toxic effect. Despite similar mechanisms of toxicity, CT is more potent than LT.

Effects of Aβ-derived peptide fragments on fibrillogenesis of Aβ.

Amyloid β (Aβ) peptide aggregation plays a central role in Alzheimer's disease (AD) etiology. AD drug candidates have included small molecules or peptides directed towards inhibition of Aβ fibrillogenesis. Although some Aβ-derived peptide fragments suppress Aβ fibril growth, comprehensive analysis of inhibitory potencies of peptide fragments along the whole Aβ sequence has not been reported.

Mutual structural effects of unmodified and pyroglutamylated amyloid β peptides during aggregation.

Amyloid β (Aβ) peptide aggregates are linked to Alzheimer's disease (AD). Posttranslationally pyroglutamylated Aβ (pEAβ) occurs in AD brains in significant quantities and is hypertoxic, but the underlying structural and aggregation properties remain poorly understood. Here, the structure and aggregation of Aβ and pEAβ are analyzed separately and in equimolar combination.

Reversal of Alpha-Synuclein Fibrillization by Protein Disulfide Isomerase.

Aggregates of α-synuclein contribute to the etiology of Parkinson's Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson's Disease.

Stability and Conformational Resilience of Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is a redox-dependent protein with oxidoreductase and chaperone activities. It is a U-shaped protein with an structural organization in which the and domains have CGHC active sites, the and domains are involved with substrate binding, and is a flexible linker. PDI exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin, suggesting PDI can regain a folded, functional conformation after exposure to stress conditions.

Quercetin-3-Rutinoside Blocks the Disassembly of Cholera Toxin by Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is mainly located in the endoplasmic reticulum (ER) but is also secreted into the bloodstream where its oxidoreductase activity is involved with thrombus formation. Quercetin-3-rutinoside (Q3R) blocks this activity, but its inhibitory mechanism against PDI is not fully understood. Here, we examined the potential inhibitory effect of Q3R on another process that requires PDI: disassembly of the multimeric cholera toxin (CT).

HSC70 and HSP90 chaperones perform complementary roles in translocation of the cholera toxin A1 subunit from the endoplasmic reticulum to the cytosol.

Cholera toxin (CT) travels by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) where the catalytic A1 subunit of CT (CTA1) dissociates from the rest of the toxin, unfolds, and moves through a membrane-spanning translocon pore to reach the cytosol. Heat shock protein 90 (HSP90) binds to the N-terminal region of CTA1 and facilitates its ER-to-cytosol export by refolding the toxin as it emerges at the cytosolic face of the ER membrane. HSP90 also refolds some endogenous cytosolic proteins as part of a foldosome complex containing heat shock cognate 71-kDa protein (HSC70) and the HSC70/HSP90-organizing protein (HOP) linker that anchors HSP90 to HSC70.

Membrane Pore Formation by Peptides Studied by Fluorescence Techniques.

Pore formation in cellular membranes by pathogen-derived proteins is a mechanism utilized by a set of microbes to exert their cytotoxic effect. On the other hand, the host cells have developed a defense mechanism to produce antimicrobial peptides to kill the pathogens by a similar, membrane perforation mechanism. Furthermore, certain endogenous proteins or peptides kill the parent cells through membrane permeabilization.

FTIR Analysis of Proteins and Protein-Membrane Interactions.

Fourier transform infrared (FTIR) spectroscopy has become one of the major techniques of structural characterization of proteins, peptides, and protein-membrane interactions. While the method does not have the capability of providing the precise, atomic-resolution molecular structure, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or intermolecular interactions. The sensitivity of vibrational frequencies to atomic masses has led to development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein.

Structure of amyloid β in lipid environment and cholesterol-dependent membrane pore formation.

The amyloid β (Aβ) peptide and its shorter variants, including a highly cytotoxic Aβ peptide, exert their neurotoxic effect during Alzheimer's disease by various mechanisms, including cellular membrane permeabilization. The intrinsic polymorphism of Aβ has prevented the identification of the molecular basis of Aβ pore formation by direct structural methods, and computational studies have led to highly divergent pore models. Here, we have employed a set of biophysical techniques to directly monitor Ca-transporting Aβ pores in lipid membranes, to quantitatively characterize pore formation, and to identify the key structural features of the pore.

Protein disulfide isomerase does not act as an unfoldase in the disassembly of cholera toxin.

Cholera toxin (CT) is composed of a disulfide-linked A1/A2 heterodimer and a ring-like, cell-binding B homopentamer. The catalytic A1 subunit must dissociate from CTA2/CTB to manifest its cellular activity. Reduction of the A1/A2 disulfide bond is required for holotoxin disassembly, but reduced CTA1 does not spontaneously separate from CTA2/CTB: protein disulfide isomerase (PDI) is responsible for displacing CTA1 from its non-covalent assembly in the CT holotoxin.

From the Wave Equation to Biomolecular Structure and Dynamics.

The multiscale models for complex chemical systems constitute a powerful computational tool to describe biomolecular structure and dynamics, including enzymatic reactions. Here, the development of this method is presented as a miraculous chain of events, involving astoundingly lucky encounters of brilliant minds such as Planck, Schrödinger, Pauling, Karplus, Levitt, and Warshel.

Membrane Binding and Pore Formation by a Cytotoxic Fragment of Amyloid β Peptide.

Amyloid β (Aβ) peptide contributes to Alzheimer's disease by a yet unidentified mechanism. In the brain tissue, Aβ occurs in various forms, including an undecapeptide Aβ, which exerts a neurotoxic effect through the mitochondrial dysfunction and/or Ca-permeable pore formation in cell membranes. This work was aimed at the biophysical characterization of membrane binding and pore formation by Aβ.

Unmodified and pyroglutamylated amyloid β peptides form hypertoxic hetero-oligomers of unique secondary structure.

Amyloid β (Aβ) peptide plays a major role in Alzheimer's disease (AD) and occurs in multiple forms, including pyroglutamylated Aβ (AβpE). Identification and characterization of the most cytotoxic Aβ species is necessary for advancement in AD diagnostics and therapeutics. While in brain tissue multiple Aβ species act in combination, structure/toxicity studies and immunotherapy trials have been focused on individual forms of Aβ.

Interfacial Enzymes: Membrane Binding, Orientation, Membrane Insertion, and Activity.

Most interfacial enzymes undergo activation upon membrane binding. Interfacial activation is determined not only by the binding strength but also by the specific mode of protein-membrane interactions, including the angular orientation and membrane insertion of the enzymes. This chapter describes biophysical techniques to quantitatively evaluate membrane binding, orientation, membrane insertion, and activity of secreted phospholipase A (PLA) and lipoxygenase (LO) enzymes.

Thermal Unfolding of the Pertussis Toxin S1 Subunit Facilitates Toxin Translocation to the Cytosol by the Mechanism of Endoplasmic Reticulum-Associated Degradation.

Pertussis toxin (PT) moves from the host cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic PTS1 subunit dissociates from the rest of the toxin in the ER and then shifts to a disordered conformation which may trigger its export to the cytosol through the quality control mechanism of ER-associated degradation (ERAD). Functional roles for toxin instability and ERAD in PTS1 translocation have not been established.

Structural Dynamics of Insulin Receptor and Transmembrane Signaling.

The insulin receptor (IR) is a (αβ)2-type transmembrane tyrosine kinase that plays a central role in cell metabolism. Each αβ heterodimer consists of an extracellular ligand-binding α-subunit and a membrane-spanning β-subunit that comprises the cytoplasmic tyrosine kinase (TK) domain and the phosphorylation sites. The α- and β-subunits are linked via a single disulfide bridge, and the (αβ)2 tetramer is formed by disulfide bonds between the α-chains.

Isotope-edited FTIR reveals distinct aggregation and structural behaviors of unmodified and pyroglutamylated amyloid β peptides.

Amyloid β peptide (Aβ) is causatively associated with Alzheimer's disease (AD), and N-terminally truncated and pyroglutamylated Aβ peptides (AβpE) exert hypertoxic effect by an unknown mechanism. Recent evidence has identified the prefibrillar oligomers of Aβ, not the fibrils, as the prevalent cytotoxic species. Structural characterization of Aβ and AβpE oligomers is therefore important for better understanding of their toxic effect.

Morphology-Dependent HIV-Enhancing Effect of Semen-Derived Enhancer of Viral Infection.

PAP248-286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248-286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248-286 is morphology dependent.

Use of the amicyanin signal sequence for efficient periplasmic expression in E. coli of a human antibody light chain variable domain.

Periplasmic localization of recombinant proteins offers advantages over cytoplasmic protein expression. In this study signal sequence of amicyanin, which is encoded by the mauC gene of Paracoccus denitrificans, was used to express the light chain variable domain of the human κIO8/O18 germline antibody in the periplasm of Escherichiacoli. The expressed protein was purified in good yield (70mg/L of culture) in one step from the periplasmic fraction by affinity chromatography using an engineered hexahistidine tag.