The protein fluorescence and structural toolkit: Database and programs for the analysis of protein fluorescence and structural data

Shen, Chien-Hua; Menon, Rajiv G.; Das, Dipanwita; Bansal, Nidhi; Nahar, Neha; Guduru, Neelima; Jaegle, Stephen; Peckham, Joan; Reshetnyak, Yana K.

doi:10.1002/prot.21857

Cited by 39 publications

(41 citation statements)

References 28 publications

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“…We previously demonstrated that the emission of tryptophan residues of pHLIP is distinct in each of the three states. In state I (in solution), the tryptophan residues of pHLIP are completely exposed to the polar and flexible water environment, resulting in long wavelength fluorescence, (Class III according to the Burstein and Reshetnyak classification (13,14). In state II (adsorbed to the bilayer surface), tryptophan residues are partially buried in the hydrophobic core of the lipid bilayer, leading to a blue shift of the emission and an increase of intensity.…”

Section: Resultsmentioning

confidence: 99%

“…At low lipid/peptide ratios the values of coordination number are lower and change from 19 to 6 with increasing temperature. We assume that each peptide helix might be surrounded by the same number of lipids (14) as in the case of high lipid/peptide ratios, but the lipids might be shared between helices (one lipid interacting with two different helices at the same time). However, we cannot exclude the possibility of some helix aggregation at low lipid/peptide ratios.…”

Section: (For 37°c) (B)mentioning

confidence: 99%

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Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Reshetnyak

Andreev

Segala

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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166

180

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The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric ␣-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I-state II transition) at 37°C is approximately ؊7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II-state III transition) is nearly ؊2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.thermodynamics ͉ drug delivery ͉ imaging ͉ membrane protein T he folding of helical membrane proteins can be conceptualized in terms of distinct steps: (i) peptide insertion and folding to form independently stable helices across a lipid bilayer, (ii) helix association to form a helix bundle intermediate, and (iii) further rearrangements of protein structure and/or binding of prosthetic groups to achieve a functional state (1). Insertion of most membrane proteins is facilitated in vivo by complex molecular machines, such as the translocon, which assist in placing hydrophobic sequences across the bilayer (2). In contrast to more hydrophobic sequences, moderately polar transmembrane domains of Cterminally-anchored proteins can postranslationally translocate themselves into membranes in a translocon-defective yeast strain (3, 4), seeking the free-energy minimum (5). Thus, insertion of a disordered peptide in aqueous solution to form a transbilayer ␣-helix is accompanied by a release of energy, so thermodynamic studies of the interaction of proteins and peptides with a lipid bilayer can provide insights regarding folding. However, such studies are significantly complicated by the poor solubility of membrane peptides, which has made it difficult to separate the contributions of peptide interactions with a lipid bilayer from self-interactions and aggregation.We have been studying a useful system for measurement of the thermodynamics and kinetics of peptide insertion and folding across a lipid bilayer. It is based on the pH low-insertion peptide (pHLIP), which has three major states: (I) soluble in water in an unstructured, monomeric state, (II) bound to the surface of a lipid bilayer in an unstructu...

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Section: Resultsmentioning

confidence: 99%

Section: (For 37°c) (B)mentioning

confidence: 99%

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Reshetnyak

Andreev

Segala

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

166

180

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“…4) support a similar polar and hydrophobic setting in AgTRPA1 for Trp-779 and Trp-950, respectively. The expected emission maximum for a tryptophan of class III is between 346 and 350 nm, whereas the ranges for tryptophans belonging to classes S and I are blue-shifted to 321-325 and 330 -333 nm (40). Concentration-response experiments show that both constructs have intrinsic tryptophan fluorescence with a maximum at 337 nm, which can be totally quenched by the agonist AITC without any associated shift of the maximum in the spectra (Fig.…”

Section: Intrinsic Tryptophan Fluorescence Ismentioning

confidence: 91%

“…3B). Two conserved tryptophans, corresponding to Trp-779 and Trp-950 in AgTRPA1, are buried in the structure: a classification of these tryptophans in the structure of hTRPA1 using PFAST (Protein Fluorescence and Structure Toolkit (40)) suggests that the former is located in a fairly polar environment (class III, p ϭ 0.87), whereas the latter is packed into more hydrophobic milieu (class S (p ϭ 0.55) or class I (p ϭ 0.45)). The conserved amino acid residues within 5 Å of the two tryptophan indole rings (Fig.…”

Section: Intrinsic Tryptophan Fluorescence Ismentioning

confidence: 99%

The N-terminal Ankyrin Repeat Domain Is Not Required for Electrophile and Heat Activation of the Purified Mosquito TRPA1 Receptor

Survery

Moparthi

Kjellbom

et al. 2016

Journal of Biological Chemistry

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Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing; some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional because they responded to the electrophilic compounds allyl isothiocyanate and cinnamaldehyde as well as heat. The proteins' similar intrinsic fluorescence properties and corresponding quenching when activated by allyl isothiocyanate or heat suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that Ag-TRPA1 is an inherent thermo-and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog, the N-terminal domain may tune the response but is not required for the activation by these stimuli.

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“…Steady state tryptophan fluorescence is a useful technique to probe the environment around the aromatic amino acid residues (33,34). Illuminating proteins with 290-nm excitation wavelength selectively excites tryptophan.…”

Section: Conformation and Hydrodynamic Radii Of Wt And Mutantmentioning

confidence: 99%

Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins

Lakshminarayanan¹,

Bromley²,

Lei

et al. 2010

Journal of Biological Chemistry

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Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25°C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25°C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations.Tooth enamel is one of the hardest and most heavily mineralized vertebrate tissues that can withstand wear without catastrophic failure during the entire life span of an organism (1, 2). The formation of enamel takes place in an extracellular environment, including an array of complex proteins and proteases in three main stages, namely the secretory, transition, and maturation stages (3). As the enamel development progresses, proteases such as enamelysin (MMP-20) and later kallikrein 4 (KLK-4) cleave the proteins, which are removed from the mineralization site in the extracellular matrix enabling the growth of enamel crystals and resulting in enamel hardness. Thus, during amelogenesis, the soft mineralized tissue formed during the early stages becomes hard and tough and almost devoid of any proteins at the maturation stage. Amelogenin is the major constituent (ϳ90%) of the protein matrix during the secretory stage and, together with other proteins in enamel, is responsible for the hierarchical structure observed in the enamel prisms (4 -6).In vitro amelogenin self-assembles into spherical structures, and this property depends on pH, ionic strength, and protein concentration (7). Using CD spectropolarimetry and NMR, we have reported that recomb...

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The protein fluorescence and structural toolkit: Database and programs for the analysis of protein fluorescence and structural data

Cited by 39 publications

References 28 publications

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

The N-terminal Ankyrin Repeat Domain Is Not Required for Electrophile and Heat Activation of the Purified Mosquito TRPA1 Receptor

Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins

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