Addition Of Trifluoroethanol Research Articles

Artificial Proteins Designed from G3LEA Contribute to Enhancement of Oxidation Tolerance in E. coli in a Chaperone-like Manner.

G3LEA is a family of proteins that exhibit chaperone-like activity when under distinct stress. In previous research, DosH was identified as a G3LEA protein from model extremophile-Deinococcus radiodurans R1 with a crucial core HD domain consisting of eight 11-mer motifs. However, the roles of motifs participating in the process of resistance to stress and their underlying mechanisms remain unclear. Here, eight different proteins with tandem repeats of the same motif were synthesized, named Motif1-8, respectively, whose function and structure were discussed. In this way, the role of each motif in the HD domain can be comprehensively analyzed, which can help in finding possibly crucial amino acid sites. Circular dichroism results showed that all proteins were intrinsically ordered in phosphate buffer, and changed into more α-helical ordered structures with the addition of trifluoroethanol and glycerol. Transformants expressing artificial proteins had significantly higher stress resistance to oxidation, desiccation, salinity and freezing compared with the control group; E. coli with Motif1 and Motif8 had more outstanding performance in particular. Moreover, enzymes and membrane protein protection viability suggested that Motif1 and Motif8 had more positive influences on various molecules, demonstrating a protective role in a chaperone-like manner. Based on these results, the artificial proteins synthesized according to the rule of 11-mer motifs have a similar function to wildtype protein. Regarding the sequence in all motifs, there are more amino acids to produce H bonds and α-helices, and more amino acids to promote interaction between proteins in Motif1 and Motif8; in addition, considering linkers, there are possibly more amino acids forming α-helix and binding substrates in these two proteins, which potentially provides some ideas for us to design potential ideal stress-response elements for synthetic biology. Therefore, the amino acid composition of the 11-mer motif and linker is likely responsible for its biological function.

Uniform Biodegradable Fiber-Like Micelles and Block Comicelles via "Living" Crystallization-Driven Self-Assembly of Poly(l-lactide) Block Copolymers: The Importance of Reducing Unimer Self-Nucleation via Hydrogen Bond Disruption.

Fiber-like micelles based on biodegradable and biocompatible polymers exhibit considerable promise for applications in nanomedicine, but until recently no convenient methods were available to prepare samples with uniform and controllable dimensions and spatial control of functionality. "Living" crystallization-driven self-assembly (CDSA) is a seeded growth method of growing importance for the preparation of uniform 1D and 2D core-shell nanoparticles from a range of crystallizable polymeric amphiphiles. However, in the case of poly(l-lactide) (PLLA), arguably the most widely utilized biodegradable polymer as the crystallizable core-forming block, the controlled formation of uniform fiber-like structures over a substantial range of lengths by "living" CDSA has been a major challenge. Herein, we demonstrate that via simple modulation of the solvent conditions via the addition of trifluoroethanol (TFE), DMSO, DMF and acetone, uniform fiber-like nanoparticles from PLLA diblock copolymers with controlled lengths up to 1 μm can be prepared. The probable mechanism involves improved unimer solvation by a reduction of hydrogen bonding interactions among PLLA chains. We provide evidence that this minimizes undesirable unimer aggregation which otherwise favors self-nucleation that competes with epitaxial crystallization from seed termini. This approach has also allowed the formation of well-defined segmented block comicelles with PLLA cores via the sequential seeded-growth of PLLA block copolymers with different corona-forming blocks.

The Role of H-Bonding in PCET: The Chemically Reversible One Electron, Three Acid Reduction of N-Methyl-4,4'-Bipyridine Radical in Acetonitrile

Oxidation and reduction of organic redox-couples typically leads to huge changes in their acidity/basicity, with the result that proton transfer (PT) often accompanies electron transfer (ET). These types of reactions are known as proton-coupled-electron-transfer or PCET reactions. While the prevalence of PCET reactions in reversible organic redox transformations is something that has been recognized for over 100 years, it is only much more recently that it has become apparent that H-bonding intermediates often play a significant role in the mechanism of these reactions. One way in which H-bonding can do so is through what we have termed a “wedge-scheme” mechanism in which ET and PT occur through the H-bond intermediate formed between the redox-active acid or base and the added base or acid.1 This can provide an easier path for ET-PT in either direction and thus increase the reversibility of the overall reaction. Another means through which H-bonding can affect PCET is by stabilizing the products. In the PCET reaction to be discussed in this presentation, reduction of N-methyl-4,4'-bipyridinium (commonly called “monoquat” or MQ) in acetonitrile in the presence of acidic alcohols, it appears that both H-bond effects are present. MQ undergoes two reversible one electron reductions in acetonitrile, the first corresponding to reduction of the starting cation, MQ+, to an uncharged radical, MQ. This is followed by a second reduction at significantly more negative potentials of the radical to the anion, MQ−, which formally contains a negatively-charged nitrogen and is thus quite basic. Not surprisingly, CV studies show that addition of trifluoroethanol or dichloroethanol, both fairly acidic alcohols, result in no change in E1/2 of the first reduction but a significant positive shift in the E1/2 of the second reduction, most likely due to protonation of the anion. The second reduction wave is slightly broad, but continues to shift positive and remain chemically reversible up to at least 400 equivalents of added alcohol, at which point it has begun to merge with the first reduction. What is surprising, in addition to the chemical reversibility, is that analysis of the observed E1/2 as a function of added alcohol concentration is consistent with a single 1e− transformation involving three alcohols throughout the entire range. Since it is highly unlikely that the MQ anion would be triply protonated, our current hypothesis is that the product conjugate base of the alcohol forms a strongly H-bonded complex with two other alcohols, making the overall reaction MQ + 3ROH + e− = MQH + (RO−)(ROH)2. The mechanism would involve initial 1 e−, 1H+ transfer occurring through the H-bond intermediate (wedge scheme), followed by rapid formation of the homoconjugate complex (RO−)(ROH)2. The results of current and future work on this system to further elucidate the mechanism, and either support or refute the wedge-scheme/homoconjugate hypothesis, will be presented. 1 L. A. Clare, A. T. Pham, F. Magdaleno, J. Acosta, J. E. Woods and D. K. Smith, Journal of the American Chemical Society 2013, 135, 18930-18941.

Exploring the Structural Properties of Synaptotagmin's Intrinsically Disordered Region

We used time-resolved FRET, circular dichroism, and all-atom simulation to investigate the structural impact of phosphorylation and dielectric constant on synaptotagmin 1's intrinsically disordered region (IDR). We found that the full-length IDR sequence, a ∼60 residue strong polyampholyte that when studied in the form of a peptide, undergoes structural collapse consistent with its κ-predicted behavior. Furthermore, we found that the exocytosis-modulating phosphorylation of Thr112, a residue located in the IDR's more sequence diverse central core region, disrupts a local disorder-to-order transition that occurs when solution dielectric constant is lowered and helical structure is stabilized by addition of trifluoroethanol. Implicit solvent simulations testing the impact of dielectric constant alone converge on a similar result, showing that helical structure is formed with higher probability at a reduced dielectric, where several lysine-aspartic acid salt bridges stabilize transient secondary structure. Phosphorylation, however, results in formation of salt bridges unsuitable for helix formation. These results suggest a model where compaction of the IDR sequence and phosphorylation may regulate structural transitions that in turn modulate neuronal exocytosis.

Joining Nanotubes Comprising Nucleobase-carrying Amphiphilic Polypeptides.

Three kinds of amphiphilic polypeptides, X-poly(sarcosine)-b-(L-Leu-Aib)6 (X = adenine, thymine, glycolic acid), were synthesized and self-assembled in a tris buffer to take on nanotube morphology. The nanotubes were joined together to extend the nanotube length with the addition of trifluoroethanol and heat treatment at 50 °C for 24 h. The length extension rate decreased in the order of adenine > glycolic acid > thymine depending on the N-terminal chromophores. Adenine-adenine interactions between the nanotubes were found to be more prevalent upon joining the nanotubes than adenine-thymine interactions. Further, adenines on the nanotube surface could chelate with Cu(<small>ii</small>) to thermodynamically stabilize the nanotube membrane. AFM imaging in liquid environment revealed that the membrane elasticity of the adenine nanotube was as high as ca. 1 MPa, which is considered to be strengthened as a result of the adenine-adenine interactions.

The Role of H-Bonding in Pcet: The Chemically Reversible One Electron, Two Proton Reduction of N-Methyl-4,4'-Bipyridine Radical in Acetonitrile

Oxidation and reduction of organic redox-couples typically leads to huge changes in their acidity/basicity, with the result that proton transfer (PT) often accompanies electron transfer (ET). These types of reactions have come to be known as proton-coupled-electron-transfer or PCET reactions. While the prevalence of PCET reactions in reversible organic redox transformations is something that has been recognized for over 100 years, it is only much more recently that it has become apparent that H-bonding intermediates often play a significant role in the mechanism of these reactions. One way in which H-bonding can do so is through what we have termed a “wedge-scheme” mechanism in which ET and PT occur through the H-bond intermediate formed between the redox-active acid or base and the added base or acid.1 This can provide an easier path for ET-PT in either direction and thus increase the reversibility of the overall reaction. Previously we have demonstrated the utility of the wedge scheme in oxidation reactions involving phenylenediamines and bases.1,2 In new work, we are exploring the utility of the wedge scheme in explaining the chemically reversible PCET reaction occurring upon addition of acidic alcohols to the N-methyl-4,4'-bipyridinium redox couple (commonly called “monoquat” or MQ) in acetonitrile. This compound undergoes two reversible one electron reductions in acetonitrile, the first corresponding to reduction of the starting cation, MQ+, to an uncharged radical, MQ. This is followed by a second reduction at significantly more negative potentials of the radical to the anion, MQ-, which formally contains a negatively-charged nitrogen and is thus quite basic. Not surprisingly, cyclic voltammetry (CV) studies show that addition of trifluoroethanol (TFE), a fairly acidic alcohol, results in no change in E1/2 of the first reduction but a significant positive shift in the E1/2 of the second reduction, most likely due to protonation of the anion. The second reduction wave is slightly broad, but continues to shift positive and remain chemically reversible up to at least 400 equivalents of added alcohol, at which point it has shifted over 0.6 V and almost merged with the first reduction wave. What is perhaps surprising, in addition to the chemical reversibility, is that analysis of the observed E1/2 as a function of added alcohol concentration is consistent with a single 1e-, 2 H+ transformation (MQ + 2 HA + 1e- = MQH2+ + 2 A-) throughout the entire range. Qualitatively similar behavior observed with addition of alcohols to quinones in acetonitrile has commonly been attributed to strong H-bonding between the alcohol and the quinone dianion. However, a H-bonding only mechanism is not likely in this case both because of the expected basicity of MQ- and the uniformity of the reaction stoichiometry over the entire concentration range. (With H-bonding only, the stoichiometry would likely start 1:1, then go to 2:1 at higher concentrations.) On the other hand, a classic step-wise PCET mechanism, ET-PT-PT, can not explain the observed reversibility over the entire concentration range. What we believe can explain all of the above is initial 1 e-, 1H+ transfer occurring through the H-bond intermediate (wedge scheme), followed by the second 1H+ transfer. The results of current and future work on this system to further elucidate the mechanism, and either support or refute the wedge-scheme hypothesis, will be described in this presentation. These will include studies with other acids, CV simulations and analysis of deuterium isotope effects 1 L. A. Clare, A. T. Pham, F. Magdaleno, J. Acosta, J. E. Woods and D. K. Smith, Journal of the American Chemical Society 2013, 135, 18930-18941. 2B. T. Tamashiro, M. R. Cedano, A. T. Pham and D. K. Smith, Journal of Physical Chemistry C 2015, 119, 12865-12874.

Aggregation Pathways of Native-Like Ubiquitin Promoted by Single-Point Mutation, Metal Ion Concentration, and Dielectric Constant of the Medium.

Ubiquitin-positive protein aggregates are biomarkers of neurodegeneration, but the molecular mechanism responsible for their formation and accumulation is still unclear. Possible aggregation pathways of human ubiquitin (hUb) promoted by both intrinsic and extrinsic factors, are here investigated. By a computational analysis, two different hUb dimers are indicated as possible precursors of amyloid-like structures, but their formation is disfavored by an electrostatic repulsion involving Glu16 and other carboxylate residues present at the dimer interface. Experimental data on the E16V mutant of hUb shows that this single-point mutation, although not affecting the overall protein conformation, promotes protein aggregation. It is sufficient to shift the same mutation by only two residues (E18V) to regain the behavior of wild-type hUb. The neutralization of Glu16 negative charge by a metal ion and a decrease of the dielectric constant of the medium by addition of trifluoroethanol (TFE), also promote hUb aggregation. The outcomes of this research have important implications for the prediction of physiological parameters that favor aggregate formation.

Conformational behavior of alpha-2-macroglobulin: Aggregation and inhibition induced by TFE

Alpha-2-macroglobulin (α2M), a pan-proteinase inhibitor, inhibits a variety of endogenous and exogenous proteinases and constitutes an important part of body’s innate defense system. In the present study, we explored how trifluoroethanol (TFE) may modulate the structure, antiproteinase activity and aggregation of α2M. TFE was sequentially added over a range of 0–20% (v/v) and the effects induced were studied by activity assay, intrinsic fluorescence, ANS fluorescence, circular dichroism, turbidity assay, Rayleigh scattering measurement and ThT fluorescence measurement. Decrease in activity and increase in fluorescence intensity of α2M upon addition of TFE shows structural deviation from the native structure and suggests aggregation of protein upon solvent addition. Increase in turbidity and Rayleigh scattering of modified α2M confirms the formation of aggregates. Insignificant ThT fluorescence intensity of TFE treated α2M is indicative of amorphous or non-amyloid aggregation. Further, circular dichroism results indicate the changes in secondary structure of native α2M as negative ellipticity decreased on addition of the polar solvent to the inhibitor. The turbidometric analysis, Rayleigh scattering, ThT fluorescence intensity of modified α2M suggests that the protein might be driven towards non-amyloid or amorphous aggregation. Our studies provide important mechanistic insight how α2M undergoes conformational and functional changes when exposed to TFE.

The Effects of Trifluoroethanol on the Helical Structure of SIKE

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure of SIKE. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To examine the structure of SIKE we have performed two dimensional proton NMR experiments and Circular Dichroism spectroscopy and compared the results with both secondary and tertiary structure predictions. 2D NOESY spectra obtained using several mixing times in general revealed little stable structure in the molecule as detected in the NH‐NH cross peak region, suggesting the protein may contain significant disordered regions. CD spectra suggest significant alpha helix content and the possibility of coiled coil regions of structure. Addition of Trifluoroethanol, known to stabilize alpha helical structures, led to increased CD signal at 222nm. Using a range of TFE concentrations optimal stabilization of helical structure in SIKE allowed comparison with helical secondary structure predications.Support or Funding InformationThis work was supported by NIH grant R21A1 107447 to JKB

Functional expression, monodispersity and conformational changes in the SBMV virus viral VPg on binding TFE

Southern bean mosaic virus (SBMV) RNA purified from infected plants was used for cloning the viral genome-linked protein (VPg) and was subsequently expressed in Escherichia coli. Circular dichroism (CD), dynamic light scattering (DLS) and saturation transfer difference (STD) by nuclear magnetic resonance (NMR) measurements were employed to determine the degree of monodispersity and to investigate the conformational changes in the absence and presence of trifluoroethanol (TFE) which indicated increased helical content with increasing concentration of TFE. 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as a probe to compare the unfolding regions of the protein before and after addition of TFE. The results indicated that although the TFE concentration influences VPg folding, it does not play a role in nucleotide binding and that the local solvent hydrophobicity causes significant conformational changes.

An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions.

During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20-48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes invitro. The structure of the NPBP/ΔNPNTD complex, solved to3.7Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.

Beaded nanofibers assembled from double-hydrophobic elastin-like block polypeptides: Effects of trifluoroethanol.

A "double-hydrophobic" elastin-like triblock polypeptide GPG has been constructed by mimicking the localization of proline- and glycine-rich hydrophobic domains of native elastin, a protein that provides elasticity and resilience to connective tissues. In this study, the effects of trifluoroethanol (TFE), an organic solvent that strongly affects secondary structures of polypeptides on self-assembly of GPG in aqueous solutions were systematically studied. Beaded nanofiber formation of GPG, where nanoparticles are initially formed by coacervation of the polypeptides followed by their connection into one-dimensional nanostructures, is accelerated by the addition of TFE at the concentrations up to 30% (v/v), whereas aggregates of nanoparticles are formed at 60% TFE. The concentration-dependent assembly pattern discussed is based on the influence of TFE on the secondary structures of GPG. Well-defined nanofibers whose diameter and secondary structures are controlled by TFE concentration may be ideal building blocks for constructing bioelastic materials in tissue engineering.

Synchrotron Radiation Circular Dichroism Spectroscopy of MEG14, an Intrinsically Disordered Protein

The micro-exon genes (MEG) identified in the genome of Schistossoma mansoni, a flatworm parasite responsible for the second most widespread tropical disease, code for a class of small secreted proteins that are highly expressed in the human host stage. Due to the high variability in primary structure of the MEG proteins, the parasite is thought to recruit them to trick the host immune system; however, their specific function is still not understood. We have examined the secondary structure of MEG14, a member of this family, as well as the effects of a range of physical-chemical factors on its conformation, using synchrotron radiation circular dichroism (SRCD) spectroscopy and Oriented-SRCD. The SRCD spectrum of MEG14 in aqueous solution exhibits a strong minimum at 198 nm and a small positive band at 182 nm, corresponding a high content of disordered structure (>80%). However, disorder-to-order transitions that lead to the induction of an alpha-helix structure in MEG14 were noted by the addition of trifluoroethanol (≥ 50%) with negative peak at 208 and 222 nm and a stronger positive peak at 192 with a shoulder at 175 nm. This ordering effect was also produced by increasing temperature (in a reversible process), the presence of negatively charged lipids (like POPG and SDS), and absorption on a silica surface, followed by dehydration. Taken together with consideration of its primary structure (high content of charged and proline residues, low content of aromatic and/or hydrophobic residues), these results support the classification of MEG14 as good model of an intrinsically disordered protein, and suggest the possibility of its interaction with different partners in the host to produce multifunctional roles.(Support by grants from FAPESP, CNPq, MCTI, and the U.K. BBRSC, and beamtime from the ANKA, ISA and Soleil Synchrotrons)

Conversion of natively unstructured α-synuclein to its α-helical conformation significantly attenuates production of reactive oxygen species

The intracellular α-synuclein (α-syn) protein, whose conformational change and aggregation have been closely linked to the pathology of Parkingson's disease (PD), is highly populated at the presynaptic termini and remains there in the α-helical conformation. In this study, circular dichroism confirmed that natively unstructured α-syn in aqueous solution was transformed to its α-helical conformation upon addition of trifluoroethanol (TFE). Electrochemical and UV–visible spectroscopic experiments reveal that both Cu (I) and Cu (II) are stabilized, with the former being stabilized by about two orders of magnitude. Compared to unstructured α-syn (Binolfi et al., J. Am. Chem. Soc. 133 (2011) 194–196), α-helical α-syn stabilizes Cu (I) by more than three orders of magnitude. Through the measurements of H2O2 and hydroxyl radicals (OH) in solutions containing different forms of Cu (II) (free and complexed by unstructured or α-helical α-syn), we demonstrate that the significantly enhanced Cu (I) binding affinity helps inhibit the production of highly toxic reactive oxygen species, especially the hydroxyl radicals. Our study provides strong evidence that, as a possible means to prevent neuronal cell damage, conversion of the natively unstructured α-syn to its α-helical conformation in vivo could significantly attenuate the copper-modulated ROS production.

Intramolecular bridges formed by photoswitchable click amino acids

Photoswitchable click amino acids (PSCaa) are amino acids bearing a side chain consisting of a photoswitchable unit elongated with a functional group that allows for a specific click reaction, such as an alkene that can react with the thiol group of a cysteine residue. An intramolecular click reaction results in the formation of a photoswitchable bridge, which can be used for controlling conformational domains in peptides and proteins. The ability to control conformations as well as the efficiency of the intramolecular bridging depends on the length of the PSCaa side chain and the distance to the cysteine residue to be clicked with. On comparing i,i+4 and i,i+7 spacings of PSCaa and cysteine in a model peptide without a preferred conformation, it was seen that the thiol–ene click reaction takes place efficiently in both cases. Upon induction of an α-helical structure by the addition of trifluoroethanol, the thiol click reaction occurs preferentially with the i,i+4 spacing. Even in the presence of glutathione as an additional thiol the click reaction of the PSCaa occurs intramolecularly with the cysteine rather than with the glutathione, indicating that the click reaction may be used even under reducing conditions occurring in living cells.

Tryptophan side chain conformers monitored by NMR and time‐resolved fluorescence spectroscopies

We have inserted a tryptophan (F77W) in the core of the regulatory domain of cardiac troponin C (cNTnC), and previously determined the structure of this mutant with and without the cosolvent trifluoroethanol (TFE). Interestingly, the orientations of the indole side chain of the Trp are in opposite directions in the two structures (Julien et al., Protein Sci 2009; 18:1165-1174). Fluorescence decay experiments for single Trp-containing proteins often show several lifetimes, which have been interpreted as reflecting conformational heterogeneity of the Trp side chain resulting from different rotamers. To test this interpretation, we monitored the effect of TFE on wild type, F77W and F77W-V82A calcium-saturated cNTnC using 2D (13)C-HSQC NMR and time-correlated single photon counting fluorescence spectroscopies. The time dependence of the Trp fluorescence decay was fit with three lifetimes. Addition of TFE caused a gradual, but limited decrease of the lifetimes due to dynamic quenching. For F77W cNTnC, the amplitude fractions of the lifetimes also changed upon addition of TFE-the long lifetime increased from 13 to 29%, while the middle lifetime decreased from 63 to 50% and the short lifetime remained relatively unchanged. For F77W-V82A cNTnC, comparable NMR changes are observed, confirming the switch in rotamer conformation, but only much smaller changes in fluorescence decay parameters were detected. These data indicate that the balance between the rotamer states can be changed without changing the lifetime amplitude fractions appreciably, and suggest that the environment(s) of the indole ring, responsible for the different lifetimes, can result from factors other than the intrinsic rotamer state of the tryptophan.

Effect of trifluoroethanol on native and acid-induced states of glucose oxidase from Aspergillus niger

We have studied the effect of trifluoroethanol (TFE) on the native (pH 7.0), acid unfolded (pH 2.6), and molten globule (pH 1.4) states of glucose oxidase (GOX) by circular dichroism and fluorescence spectroscopy. In the presence of 50% TFE, at pH 7.0 and 2.6, GOX exhibited a transition from native coiled-coil and acid unfolded state to non-associating alpha-helical state. Interestingly, at pH 1.4, 15% TFE induced the formation of beta-structured intermediate by loss of 1-anilino-8-naphthalenesulfonate binding site and almost all tertiary contacts. The beta-structured intermediate converted into open helical conformation on further addition of TFE.

Effect of Trifluoroethanol on the Tryptophan Side Chain Orientation in the Hydrophobic Core of Troponin C Studied by NMR and Time Resolved Fluorescence

Several NMR studies have shown that co-solvent trifluoroethanol (TFE) does not perturb the overall three-dimensional structure of proteins. We have inserted a single tryptophan (F77W) in the hydrophobic core of the N-domain of cardiac troponin C (cNTnC), and determined the structure of the mutant with and without TFE (Julien et al., Protein Sci. 2009 18:1165-74). Interestingly, the position of the tryptophan side chain orientation was shown to be in opposite directions. We have monitored the effect of TFE on the tryptophan rotamer population using 13C-HSQC spectra and used a full lineshape analysis to quantify the rate of the conformational exchange.To further characterize this phenomenon, we have used Time Correlated Single Photon Counting experiments as a function of the TFE concentration. The time dependence could be fitted very well with three lifetimes in the wavelength region from 320 to 380 nm, and global analysis was further used. Addition of TFE (up to 19%) causes a gradual decrease of the lifetimes, due to dynamic quenching with very low quenching constants between kq = 0.1 to 0.01 M-1 ns-1. The amplitude fractions of the lifetimes change upon addition of TFE. At 340 nm, the amplitude fraction of the long lifetime (5.9 ns) increased from 13 to 29%, while that of the middle lifetime (3.3 ns) decreased from 63 to 50%. The short lifetime changed only to a limited extend. These data indicate that the change in the tryptophan indole position (different rotameric state) upon addition of TFE, as observed in NMR, is reflected mainly in the amplitude fractions of the different lifetimes. This is consistent, in this case, with the interpretation of lifetimes linked to rotameric states of tryptophan.

Characterization of the Structure and Intermolecular Interactions between the Connexin40 and Connexin43 Carboxyl-terminal and Cytoplasmic Loop Domains

Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels. The mechanism of heteromeric channel regulation has not been clearly defined. Tissue ischemia leads to intracellular acidification and closure of Cx43 and Cx40 homomeric channels. However, coexpression of Cx40 and Cx43 in Xenopus oocytes enhances the pH sensitivity of the channel. This phenomenon requires the carboxyl-terminal (CT) part of both connexins. In this study we used different biophysical methods to determine the structure of the Cx40CT and characterize the Cx40CT/Cx43CT interaction. Our results revealed that the Cx40CT is an intrinsically disordered protein similar to the Cx43CT and that the Cx40CT and Cx43CT can interact. Additionally, we have identified an interaction between the Cx40CT and the cytoplasmic loop of Cx40 as well as between the Cx40CT and the cytoplasmic loop of Cx43 (and vice versa). Our studies support the "particle-receptor" model for pH gating of Cx40 and Cx43 gap junction channels and suggest that interactions between cytoplasmic regulatory domains (both homo- and hetero-connexin) could be important for the regulation of heteromeric channels.

Structurally Distinct Amyloid Protofibrils Form on Separate Pathways of Aggregation of a Small Protein

Understanding the structural as well as mechanistic basis of the conformational polymorphism evident during amyloid protofibril and fibril formation by proteins is an important goal in the study of protein aggregation. In this report, we compare two separate routes to amyloid protofibril formation by the small protein barstar, one induced by the addition of trifluoroethanol (TFE) and the other by heat. The study reveals that the TFE-induced aggregation of barstar leads to protofibrils that differ from heat-induced protofibrils in their external dimensions and internal structures as well as in the mechanisms of their formation. Atomic force microscopy reveals that the TFE-induced protofibrils have about half the thickness of the heat-induced protofibrils. The thickness of the TFE-induced protofibrils (1.14 +/- 0.24) suggests that they form a beta-sheet monolayer, while the thickness of the heat-induced protofibrils (2.56 +/- 0.32) suggests that they are built up from a pair (bilayer) of beta-sheets. Fourier-transform infrared (FTIR) as well as circular dichroism (CD) spectroscopy shows that the heat-induced protofibrils are not pure beta-sheet structures but that they also contain other structures (alpha-helix and/or random coil). In contrast, the TFE-induced protofibrils contain more beta-sheet structures and less of other structures, if any. The FTIR and CD spectra also reveal that the two differently created protofibrils differ in the internal structures of their beta-sheets. The TFE-induced protofibrils differ from the heat-induced protofibrils also in the kinetics of their formation. For the heat-induced reaction, the kinetics are monophasic without any lag phase, while the kinetics of the formation of TFE-induced protofibrils are sigmoidal with an initial lag phase. It appears that the TFE-induced and the heat-induced reactions involve distinct pathways for the formation of amyloid protofibrils. The existence of alternative pathways leading to amyloid protofibrils of distinct structures has important implications in understanding the kinetic origin of amyloid polymorphism.