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To obtain proper insight into how structure develops during a protein folding reaction, it is necessary to understand the nature and mechanism of the polypeptide chain collapse reaction, which marks the initiation of folding. Here, the time-resolved fluorescence resonance energy transfer technique, in which the decay of the fluorescence light intensity with time is used to determine the time evolution of the distribution of intra-molecular distances, has been utilized to study the folding of the small protein, monellin. It is seen that when folding begins, about one-third of the protein molecules collapse into a molten globule state (I MG), from which they relax by continuous further contraction to transit to the native state. The larger fraction gets trapped into a metastable misfolded state. Exit from this metastable state occurs via collapse to the lower free energy I MG state. This exit is slow, on a time scale of seconds, because of activation energy barriers. The trapped misfolded molecules as well as the I MG molecules contract continuously and slowly as structure develops. A phenomenological model of Markovian evolution of the polymer chain undergoing folding, incorporating these features, has been developed, which fits well the experimentally observed time evolution of distance distributions. The observation that the "wrong turn" to the misfolded state has not been eliminated by evolution belies the common belief that the folding of functional protein sequences is very different from that of a random heteropolymer, and the former have been selected by evolution to fold quickly. Unlabelled Image [ABSTRACT FROM AUTHOR]

Dysregulation of tumor necrosis factor (TNF) receptor signaling is a key feature of various inflammatory disorders. Current treatments for TNF-related diseases function either by sequestering ligand or blocking ligand–receptor interactions, which can cause dangerous side effects by inhibiting the receptors that are not involved in the disease condition. Thus, alternate strategies that target receptor–receptor interactions are needed. We hypothesized that the soluble extracellular domain (ECD) of long isoform of death receptor 5 (DR5) could block endogenous receptor assembly, mimicking the biological effect of decoy receptors that lack the death domain to trigger apoptosis. Using live-cell fluorescence resonance energy transfer studies, we demonstrated that soluble ECD disrupts endogenous DR5–DR5 interactions. Cell viability assays were used to demonstrate the complete inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the ECD, although TRAIL is still able to bind to the receptor. Importantly, we used mutagenesis to prove that the inhibition of TRAIL-induced apoptosis by the ECD predominantly comes from the disruption of DR5 oligomerization and not ligand sequestration. Inhibition of death receptor activation should have important therapeutic applications in diseases such as nonalcoholic fatty liver disease. More generally, this approach should be generalized to enable the inhibition of other TNF receptor signaling mechanisms that are associated in a wide range of clinical conditions. [ABSTRACT FROM AUTHOR]

Abstract: The rate of folding of globular proteins depends on specific local and nonlocal intramolecular interactions. What is the relative role of these two types of interaction at the initiation of refolding? We address this question by application of a “double kinetics” method based on fast initiation of refolding of site specifically labeled protein samples and detection of the transient distributions of selected intramolecular distances by means of fast measurements of time‐resolved fluorescence resonance energy transfer. We determined the distribution of the distance between the ends of a 44‐chain segment that includes the AMPbind domain, by labeling residues 28 and 71, in Escherichia coli adenylate kinase (AK) and the distribution of the distance between residues 18 and 203, which depends on the overall order of the molecule. That distribution shows two-state transition to the native intramolecular distance at the same rate as that of the cooperative refolding transition of the AK molecule. In sharp contrast, the distance distribution between residues 28 and 71 is already native like at the end of the dead-time of the mixing device. This fast formation of native short distance between two widely separated chain sections can be either dependent on fast folding of the AMPbind domain or a result of a very effective nonlocal interaction between specific short clusters of hydrophobic residues. Further experiments on studying the kinetics of folding of selected structural elements in the protein will help determination of the driving force of this early folding event. [Copyright &y& Elsevier]

Abstract: The α-synuclein (αS) molecule, a polypeptide of 140 residues, is an intrinsically disordered protein that is involved in the onset of Parkinson''s disease. We applied time-resolved excitation energy transfer measurements in search of specific deviations from the disordered state in segments of the αS backbone that might be involved in the initiation of aggregation. Since at higher temperatures, the αS molecule undergoes accelerated aggregation, we studied the temperature dependence of the distributions of intramolecular segmental end-to-end distances and their fast fluctuations in eight labeled chain segments of the αS molecule. Over the temperature range of 5–40 °C, no temperature-induced unfolding or folding was detected at the N-terminal domain (residues 1–66) of the αS molecule. The intramolecular diffusion coefficient of the segments'' ends relative to each other increased monotonously with temperature. A common very high upper limiting value of ∼25 A2/ns was reached at 40 °C, another indication of a fully disordered state. Three exceptions were two segments with reduced values of the diffusion coefficients (the shortest segment where the excluded volume effect is dominant and the segment labeled in the NAC domain) and a nonlinear cooperative transition in the N-terminal segment. These specific subtle deviations from the common pattern of temperature dependence reflect specific structural constraints that could be critical in controlling the stability of the soluble monomer, or for its aggregation. Such very weak effects might be dominant in determination of the fate of ensembles of disordered polypeptides either to folding or to misfolding. [Copyright &y& Elsevier]

Abstract: We have developed a set of high-throughput screening (HTS)-compatible assays capable of measuring regulated, target-specific posttranslational modifications in a mammalian cell-based format. We chose the NFκB signal transduction cascade as a model system to validate this approach because specific target proteins in this signaling pathway undergo a multitude of posttranslational modifications in response to pathway stimulation. In this pathway, TNFα induces the phosphorylation, ubiquitination, and proteasomal degradation of IκBα, which leads to the release and translocation of the NFκB transcriptional complex into the nucleus. To measure these cellular processes, we describe the use of a stable cell line expressing a fusion of green fluorescent protein (GFP) with IκBα that can be interrogated for either ubiquitination or phosphorylation using a unique set of terbium-labeled antibodies in a time-resolved Förster resonance energy transfer (TR–FRET)-based readout. Concurrently, we have engineered a β-lactamase–IκBα reporter cell line that can be used to quantify proteasomal degradation of IκBα in living cells. Both TR–FRET and β-lactamase reporter technologies provide a convenient, sensitive, and robust means to interrogate the chronological steps in NFκB signaling in a physiologically relevant cellular context without the need to overexpress any enzyme involved in this pathway. Cellular HTS assays that interrogate such processes will provide a unique integrated approach to dissecting intermediate steps in NFκB activation and could serve as examples of broadly applicable pathway analysis tools for target-based drug discovery. [Copyright &y& Elsevier]

Abstract: Time- and space-correlated single photon counting method has been used to demonstrate the interactions of cation channel “transient receptor potential vanilloid 4” (TRPV4) and microfilaments. Living cells co-expressing TRPV4-CFP and actin-YFP, when excited for the donor molecules (CFP) exhibited an emission peak at 527nm and decrease of the lifetime in the wavelength band 460–490nm; corresponding to resonance energy transfer to YFP. CFP fluorescence decay was fitted best by a dual mode decay model. Considering the average lifetime of the donor, both in the presence and absence of acceptor yielded an apparent FRET efficiency of ∼20%. This is rather high placing the minimum distance of chromophores in the two fluorescent proteins in the range of 4nm. Thus, this study shows for the first time that TRPV4 and actin intimately associate within living cells. The significance of this finding for cell volume regulation is highlighted. [Copyright &y& Elsevier]

The Escherichia coli single-stranded DNA binding protein (SSB) binds selectively to single-stranded (ss) DNA intermediates during DNA replication, recombination and repair. Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site, thus the protein can bind to ssDNA in multiple binding modes, one of which is the (SSB)65 mode, in which a 65 nucleotide stretch of ssDNA interacts with and wraps around all four subunits of the tetramer. Previous stopped-flow kinetic studies of (SSB)65 complex formation using the oligodeoxynucleotide, (dT)70, were unable to resolve the initial binding step from the rapid wrapping of ssDNA around the tetramer. Here we report a laser temperature-jump study with resolution in the ∼500ns to 4ms time range, which directly detects these ssDNA wrapping/unwrapping steps. Biphasic time courses are observed with a fast phase that is concentration-independent and which occurs on a time-scale of tens of microseconds, reflecting the wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the slower binding phase, in combination with equilibrium binding and stopped-flow kinetic studies, also provides evidence for a previously undetected intermediate along the pathway to forming the (SSB)65 complex. [Copyright &y& Elsevier]