Visualizing the elusive open shape of G-actin in solution by SAXS data analysis

Sagar, Amin; Peddada, Nagesh; Solanki, Ashish K.; Choudhary, Vikas; Garg, Renu; Ashish, .

doi:10.1016/j.bbrc.2013.05.055

Cited by 8 publications

(17 citation statements)

References 22 publications

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“…The peak around 0.1 Å −1 and the minimum in the q range of 0.2–0.4 Å −1 indicate the presence of the helix-rich globular protein, whereas a slope lacking maximum and minimum in the middle q range suggests the existence of a random coil (Figure S2). 33,34 As shown in Figure 4, although the sequence 1 displays certain helicity, the lack of defined bell shape suggests the sequence is at least partially unfolded in solution, probably due to its short length. All other sequences adopt well-defined helical structures in solution, since they all show characteristic maximum around 0.1 Å −1 and minimum in the q range of 0.2–0.4 Å −1 .…”

Section: Resultsmentioning

confidence: 99%

“…This is because these sequences are a new class of heterogeneous peptidomimetics, CD (circular dichroism) may provide ambiguous results, since the backbone is different from regular α-peptides . On the other hand, SAXS emerges to be an excellent technique to quickly assess the solution structures of peptides or peptidomimetics. ,,,, Herein we used a Kratky plot to analyze the helicity of the lead 1:1 α/sulfono-γ-AA peptide sequences 1 , 3 , 4 , 5 , and 6 . The peak around 0.1 Å –1 and the minimum in the q range of 0.2–0.4 Å –1 indicate the presence of the helix-rich globular protein, whereas a slope lacking maximum and minimum in the middle q range suggests the existence of a random coil (Figure S2).…”

Section: Resultsmentioning

confidence: 99%

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Helical 1:1 α/Sulfono-γ-AA Heterogeneous Peptides with Antibacterial Activity

et al. 2016

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As one of the greatest threats facing the 21st century, antibiotic resistance is now a major public health concern. Host-defense peptides (HDPs) offer an alternative approach to combat emerging multi-drug-resistant bacteria. It is known that helical HDPs such as magainin 2 and its analogs adopt cationic amphipathic conformations upon interaction with bacterial membranes, leading to membrane disruption and subsequent bacterial cell death. We have previously shown that amphipathic sulfono-γ-AApeptides could mimic magainin 2 and exhibit bactericidal activity. In this article, we demonstrate for the first time that amphipathic helical 1:1 α/sulfono-γ-AA heterogeneous peptides, in which regular amino acids and sulfono-γ-AApeptide building blocks are alternatively present in a 1:1 pattern, display potent antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens. Small angle X-ray scattering (SAXS) suggests that the lead sequences adopt defined helical structures. The subsequent studies including fluorescence microscopy and time-kill experiments indicate that these hybrid peptides exert antimicrobial activity by mimicking the mechanism of HDPs. Our findings may lead to the development of HDP-mimicking antimicrobial peptidomimetics that combat drug-resistant bacterial pathogens. In addition, our results also demonstrate the effective design of a new class of helical foldamer, which could be employed to interrogate other important biological targets such as protein–protein interactions in the future.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Helical 1:1 α/Sulfono-γ-AA Heterogeneous Peptides with Antibacterial Activity

et al. 2016

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“…Finally, the G-actin was obtained by centrifugation at 40,000 ϫ g for 2 h, and the concentration was determined by measuring the absorbance at 290 nm (A 290 ϳ0.62, 1 mg/ml). The purified G-actin has been characterized recently in ATP/ADP/ AMP-NP state by SAXS analyses (27). The G-actin was labeled with a 7-fold molar excess of pyrenyl-iodoacetamide as described previously (28) and stored at Ϫ80°C for further use.…”

Section: Cloning and Purification Of Recombinant Gelsolin Mutants-mentioning

confidence: 99%

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

Peddada¹,

Sagar²,

Rathore³

et al. 2013

Journal of Biological Chemistry

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Background: Shape-function studies are necessary to design better therapeutic alternatives of the plasma gelsolin. Results: N-terminal fragment 30 -161 is the smallest segment with F-actin depolymerization potential, and G1-G3 can function independent of Ca 2ϩ ions or low pH. Conclusion: The g2-g3 linker plays a role in imparting pH/Ca 2ϩ insensitivity to G1-G3. Significance: We provide the first evidence that g2-g3 linker regulates mobility of the G1 domain.

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“…The result underpins the assignment of D T to the tumbling motion of G-actin because a sphere approximation of D T = k B T /8π ηR 3 for rotational diffusion yields a radius R of 2.7 and 2.6 nm for R6G-OEG1-G-actin and R6G-OEG3-G-actin, respectively, where k B , T , η , and R are the Boltzmann constant, temperature, viscosity, and sphere radius, respectively; these R values would be comparable to the expected radius of hydrated G-actin, as actin monomer had dimensions of 55 Å×55 Å×35 Å [ 18 ]. Additionally, a study of G-actin by small angle X-ray scattering reported values of 2.23 to 2.37 nm for the radius of gyration [ 42 ]. Importantly, Figure 7 shows that the values of both D W and ζ increase as the OEG linker length increases.…”

Section: Resultsmentioning

confidence: 99%

Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy

2015

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Investigation of the rotational motion of a fluorescent probe tethered to a protein helps to elucidate the local properties of the solvent and protein near the conjugation site of the probe. In this study, we have developed an instrument for frequency-domain fluorescence (FDF) anisotropy measurements, and studied how the local properties around a protein, actin, can be elucidated from the rotational motion of a dye tethered to actin. Rhodamine 6G (R6G) was attached to Cys-374 using newly-synthesized R6G-maleimide with three different oligo(ethylene glycol) (OEG) linker lengths. The time-resolved anisotropy decay of R6G tethered to G-actin was revealed to be a combination of the two modes of the wobbling motion of R6G and the tumbling motion of G-actin. The rotational diffusion coefficient (RDC) of R6G wobbling was ~0.1 ns−1 at 20°C and increased with OEG linker length. The use of the three R6G-actin conjugates of different linker lengths was useful to not only figure out the linker length dependence of the rotational motion of R6G but also validate the analyses. In the presence of a cosolvent of glycerol, although the tumbling motion of G-actin was retarded in response to the bulk viscosity, the wobbling motion of R6G tethered to actin exhibited an increase of RDC as glycerol concentration increased. This finding suggests an intricate relationship between the fluid properties of the bulk solvent and the local environment around actin.

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Visualizing the elusive open shape of G-actin in solution by SAXS data analysis

Cited by 8 publications

References 22 publications

Helical 1:1 α/Sulfono-γ-AA Heterogeneous Peptides with Antibacterial Activity

Helical 1:1 α/Sulfono-γ-AA Heterogeneous Peptides with Antibacterial Activity

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy

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