Physical Properties of Polymorphic Yeast Prion Amyloid Fibers

Carlos E. Castro, Jijun Dong, Mary C. Boyce, Susan Lindquist, and Matthew J. Lang


Amyloid fibers play important roles in many human diseases and natural biological processes and have immense potential as novel nanomaterials. We explore the physical properties of polymorphic amyloid fibers formed by yeast prion protein Sup35. Amyloid fibers that conferred distinct prion phenotypes ([PSI⁺]), strong (S) versus weak (W) nonsense suppression, displayed different physical properties. Both S[PSI⁺] and W[PSI⁺] fibers contained structural inhomogeneities, specifically local regions of static curvature in S[PSI⁺] fibers and kinks and self-cross-linking in W[PSI⁺] fibers. Force-extension experiments with optical tweezers revealed persistence lengths of 1.5 μm and 3.3 μm and axial stiffness of 5600 pN and 9100 pN for S[PSI⁺] and W[PSI⁺] fibers, respectively. Thermal fluctuation analysis confirmed the twofold difference in persistence length between S[PSI⁺] and W[PSI⁺] fibers and revealed a torsional stiffness of kinks and cross-links of 100–200 pN·nm/rad.


DOI


Journal: Biophysical Journal

RNA Mimics of Green Fluorescent Protein

Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey



Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.


DOI


Journal: Science

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

Katherine H. White, Marek Orzechowski, Dominique Fourmy, and Koen Visscher


Using mechanical unfolding by optical tweezers (OT) and steered molecular dynamics (SMD) simulations, we have demonstrated the critical role of Mg2+ ions for the resistance of the Beet Western Yellow Virus (BWYV) pseudoknot (PK) to unfolding. The two techniques were found to be complementary, providing information at different levels of molecular scale. Findings from the OT experiments indicated a critical role of stem 1 for unfolding of the PK, which was confirmed in the SMD simulations. The unfolding pathways of wild type and mutant appeared to depend upon pH and nucleotide sequence. SMD simulations support the notion that the stability of stem 1 is critical for −1 frameshifting. The all-atom scale nature of the SMD enabled clarification of the precise role of two Mg2+ ions, Mg45 and Mg52, as identified in the BWYV X-ray crystallography structure, in −1 frameshifting. On the basis of simulations with “partially” and “fully” hydrated Mg2+ ions, two possible mechanisms of stabilizing stem 1 are proposed. In both these cases Mg2+ ions play a critical role in stabilizing stem 1, either by directly forming a salt bridge between the strands of stem 1 or by stabilizing parallel orientation of the strands in stem 1, respectively. These findings explain the unexpected drop in frameshifting efficiency to null levels of the C8U mutant in a manner consistent with experimental observations.


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Journal: Journal of the American Chemical Society

Calcium modulates force sensing by the von Willebrand factor A2 domain

Arjen J. Jakobi, Alireza Mashaghi, Sander J. Tans, and Eric G. Huizinga



von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. VWF potency critically depends on multimer size, which is regulated by a feedback mechanism involving shear-induced unfolding of the VWF-A2 domain and cleavage by the metalloprotease ADAMTS-13. Here we report crystallographic and single-molecule optical tweezers data on VWF-A2 providing mechanistic insight into calcium-mediated stabilization of the native conformation that protects A2 from cleavage by ADAMTS-13. Unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate with non-native calcium coordination. Calcium further accelerates refolding markedly, in particular, under applied load. We propose that calcium improves force sensing by allowing reversible force switching under physiologically relevant hydrodynamic conditions. Our data show for the first time the relevance of metal coordination for mechanical properties of a protein involved in mechanosensing.


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Journal: Nature Communications

Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry

Alexxai V. Kravitz, Benjamin S. Freeze, Philip R. L. Parker, Kenneth Kay, Myo T. Thwin, Karl Deisseroth, and Anatol C. Kreitzer


Neural circuits of the basal ganglia are critical for motor planning and action selection. Two parallel basal ganglia pathways have been described, and have been proposed to exert opposing influences on motor function. According to this classical model, activation of the ‘direct’ pathway facilitates movement and activation of the ‘indirect’ pathway inhibits movement. However, more recent anatomical and functional evidence has called into question the validity of this hypothesis. Because this model has never been empirically tested, the specific function of these circuits in behaving animals remains unknown. Here we report direct activation of basal ganglia circuitry in vivo, using optogenetic control of direct- and indirect-pathway medium spiny projection neurons (MSNs), achieved through Cre-dependent viral expression of channelrhodopsin-2 in the striatum of bacterial artificial chromosome transgenic mice expressing Cre recombinase under control of regulatory elements for the dopamine D1 or D2 receptor. Bilateral excitation of indirect-pathway MSNs elicited a parkinsonian state, distinguished by increased freezing, bradykinesia and decreased locomotor initiations. In contrast, activation of direct-pathway MSNs reduced freezing and increased locomotion. In a mouse model of Parkinson’s disease, direct-pathway activation completely rescued deficits in freezing, bradykinesia and locomotor initiation. Taken together, our findings establish a critical role for basal ganglia circuitry in the bidirectional regulation of motor behaviour and indicate that modulation of direct-pathway circuitry may represent an effective therapeutic strategy for ameliorating parkinsonian motor deficits.


DOI

Journal: Nature

The INAD Scaffold Is a Dynamic, Redox-Regulated Modulator of Signaling in the Drosophila Eye

Wei Liu, Wenyu Wen,Zhiyi Wei,Jiang Yu, Fei Ye, Che-Hsiung Liu, Roger C. Hardie, and Mingjie Zhang


INAD is a scaffolding protein that regulates signaling in Drosophila photoreceptors. One of its PDZ domains, PDZ5, cycles between reduced and oxidized forms in response to light, but it is unclear how light affects its redox potential. Through biochemical and structural studies, we show that the redox potential of PDZ5 is allosterically regulated by its interaction with another INAD domain, PDZ4. Whereas isolated PDZ5 is stable in the oxidized state, formation of a PDZ45 “supramodule” locks PDZ5 in the reduced state by raising the redox potential of its Cys606/Cys645 disulfide bond by ∼330 mV. Acidification, potentially mediated via light and PLCβ-mediated hydrolysis of PIP2, disrupts the interaction between PDZ4 and PDZ5, leading to PDZ5 oxidation and dissociation from the TRP Ca2+ channel, a key component of fly visual signaling. These results show that scaffolding proteins can actively modulate the intrinsic redox potentials of their disulfide bonds to exert regulatory roles in signaling.

DOI

Journal: Cell

Proton transfer via a transient linear water-molecule chain in a membrane protein

Erik Freier, Steffen Wolf, and Klaus Gerwert



High-resolution protein ground-state structures of proton pumps and channels have revealed internal protein-bound water molecules. Their possible active involvement in protein function has recently come into focus. An illustration of the formation of a protonated protein-bound water cluster that is actively involved in proton transfer was described for the membrane protein bacteriorhodopsin (bR) [Garczarek F, Gerwert K (2006) Nature 439:109–112]. Here we show through a combination of time-resolved FTIR spectroscopy and molecular dynamics simulations that three protein-bound water molecules are rearranged by a protein conformational change that resulted in a transient Grotthuss-type proton-transfer chain extending through a hydrophobic protein region of bR. This transient linear water chain facilitates proton transfer at an intermediate conformation only, thereby directing proton transfer within the protein. The rearrangement of protein-bound water molecules that we describe, from inactive positions in the ground state to an active chain in an intermediate state, appears to be energetically favored relative to transient incorporation of water molecules from the bulk. Our discovery provides insight into proton-transfer mechanisms through hydrophobic core regions of ubiquitous membrane spanning proteins such as G-protein coupled receptors or cytochrome C oxidases.

DOI

Journal: Proceedings of the National Academy of Sciences