Visualizing Heterogeneous Protein Conformations with Multi-Tilt Nanoparticle-Aided Cryo-Electron Microscopy Sampling

Kim, Changin; Kim, Yeeun; Lee, Sang Jin; Yun, So Ri; Choi, Jungkweon; Kim, Seong Ok; Yang, Yongsoo; Ihee, Hyotcherl

doi:10.1021/acs.nanolett.3c00313

Cited by 3 publications

(3 citation statements)

References 62 publications

(140 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The topology and morphological appearances of phase-separated oligomeric states can partially be visualized using atomic force microscopy (AFM) and electron microscopy (EM). , However, detailed residue-specific changes including the structural and molecular rearrangement are still challenging even by engaging advanced structural biology tools such as NMR and single-particle cryo-EM analysis. − In this article, using Raman spectroscopy, we delineate the crucial conformation state and the status of some of the important residues in the processes of fibril formation, and we considered bovine insulin (BI) as the model globular protein for the investigation of residue-specific changes and variation of Raman marker bands.…”

Section: Introductionmentioning

confidence: 99%

Raman Spectroscopic Insights of Phase-Separated Insulin Aggregates

Dolui,

Roy,

Pal

et al. 2024

ACS Phys. Chem Au

View full text Add to dashboard Cite

Phase-separated protein accumulation through the formation of several aggregate species is linked to the pathology of several human disorders and diseases. Our current investigation envisaged detailed Raman signature and structural intricacy of bovine insulin in its various forms of aggregates produced in situ at an elevated temperature (60 °C). The amide I band in the Raman spectrum of the protein in its native-like conformation appeared at 1655 cm −1 and indicated the presence of a high content of αhelical structure as prepared freshly in acidic pH. The disorder content (turn and coils) also was predominately present in both the monomeric and oligomeric states and was confirmed by the presence shoulder amide I maker band at ∼1680 cm −1 . However, the band shifted to ∼1671 cm −1 upon the transformation of the protein solution into fibrillar aggregates as produced for a longer time of incubation. The protein, however, maintained most of its helical conformation in the oligomeric phase; the low-frequency backbone α-helical conformation signal at ∼935 cm −1 was similar to that of freshly prepared aqueous protein solution enriched in helical conformation. The peak intensity was significantly weak in the fibrillar aggregates, and it appeared as a good Raman signature to follow the phase separation and the aggregation behavior of insulin and similar other proteins. Tyrosine phenoxy moieties in the protein may maintained its H-bond donor−acceptor integrity throughout the course of fibril formation; however, it entered in more hydrophobic environment in its journey of fibril formation. In addition, it was noticed that oligomeric bovine insulin maintained the orientation/conformation of the disulfide bonds. However, in the fibrillar state, the disulfide linkages became more strained and preferred to maintain a single conformation state.

show abstract

Section: Introductionmentioning

confidence: 99%

Raman Spectroscopic Insights of Phase-Separated Insulin Aggregates

Dolui,

Roy,

Pal

et al. 2024

ACS Phys. Chem Au

View full text Add to dashboard Cite

show abstract

“…Furthermore, Aβ(1–42):CaM complexes can also function as intracellular transducers for focalized actions of Aβ peptides due to the many roles of CaM in Ca 2+ -signaling pathways modulating neuronal metabolism, excitability, and cell death known to be altered in AD [ 50 ]. The complexation between CaM and Aβ peptides is driven by interactions involving exposed hydrophobic domains of CaM and hydrophobic amino acids of Aβ [ 45 , 51 ] and produces structural changes in CaM [ 52 ].…”

Section: Introductionmentioning

confidence: 99%

Brain Hydrophobic Peptides Antagonists of Neurotoxic Amyloid β Peptide Monomers/Oligomers–Protein Interactions

Gutierrez-Merino

2023

IJMS

View full text Add to dashboard Cite

Amyloid β (Aβ) oligomers have been linked to Alzheimer’s disease (AD) pathogenesis and are the main neurotoxic forms of Aβ. This review focuses on the following: (i) the Aβ(1–42):calmodulin interface as a model for the design of antagonist Aβ peptides and its limitations; (ii) proteolytic degradation as the major source of highly hydrophobic peptides in brain cells; and (iii) brain peptides that have been experimentally demonstrated to bind to Aβ monomers or oligomers, Aβ fibrils, or Aβ plaques. It is highlighted that the hydrophobic amino acid residues of the COOH-terminal segment of Aβ(1–42) play a key role in its interaction with intracellular protein partners linked to its neurotoxicity. The major source of highly hydrophobic endogenous peptides of 8–10 amino acids in neurons is the proteasome activity. Many canonical antigen peptides bound to the major histocompatibility complex class 1 are of this type. These highly hydrophobic peptides bind to Aβ and are likely to be efficient antagonists of the binding of Aβ monomers/oligomers concentrations in the nanomolar range with intracellular proteins. Also, their complexation with Aβ will protect them against endopeptidases, suggesting a putative chaperon-like physiological function for Aβ that has been overlooked until now. Remarkably, the hydrophobic amino acid residues of Aβ responsible for the binding of several neuropeptides partially overlap with those playing a key role in its interaction with intracellular protein partners that mediates its neurotoxicity. Therefore, these latter neuropeptides are also potential candidates to antagonize Aβ peptides binding to target proteins. In conclusion, the analysis performed in this review points out that hydrophobic endogenous brain neuropeptides could be valuable biomarkers to evaluate the risk of the onset of sporadic AD, as well as for the prognosis of AD.

show abstract

“…To overcome these limitations, a myriad of cutting-edge techniques has been proposed in the fields of XRD [23][24][25], cryo-electron microscopy (cryo-EM) [26][27][28][29][30][31][32], NMR [10,22,33,34], and optical spectroscopy [35][36][37][38][39]. Notably, single-molecule spectroscopy and nanoparticleassisted cryo-electron microscopy sampling (NACS) [27,32] have emerged as viable strategies for capturing single-molecule structures, each presenting distinct benefits and limitations. Single-molecule spectroscopy is invaluable for capturing molecular behaviors and interactions at the individual molecule level, thus shedding light on the complex and dynamic processes of biomolecules.…”

Section: Introductionmentioning

confidence: 99%

Single-Molecule X-ray Scattering Used to Visualize the Conformation Distribution of Biological Molecules via Single-Object Scattering Sampling

Lee,

Ki,

Lee

et al. 2023

IJMS

Self Cite

View full text Add to dashboard Cite

Biological macromolecules, the fundamental building blocks of life, exhibit dynamic structures in their natural environment. Traditional structure determination techniques often oversimplify these multifarious conformational spectra by capturing only ensemble- and time-averaged molecular structures. Addressing this gap, in this work, we extend the application of the single-object scattering sampling (SOSS) method to diverse biological molecules, including RNAs and proteins. Our approach, referred to as “Bio-SOSS”, leverages ultrashort X-ray pulses to capture instantaneous structures. In Bio-SOSS, we employ two gold nanoparticles (AuNPs) as labels, which provide strong contrast in the X-ray scattering signal, to ensure precise distance determinations between labeled sites. We generated hypothetical Bio-SOSS images for RNAs, proteins, and an RNA–protein complex, each labeled with two AuNPs at specified positions. Subsequently, to validate the accuracy of Bio-SOSS, we extracted distances between these nanoparticle labels from the images and compared them with the actual values used to generate the Bio-SOSS images. Specifically, for a representative RNA (1KXK), the standard deviation in distance discrepancies between molecular dynamics snapshots and Bio-SOSS retrievals was found to be optimally around 0.2 Å, typically within 1 Å under practical experimental conditions at state-of-the-art X-ray free-electron laser facilities. Furthermore, we conducted an in-depth analysis of how various experimental factors, such as AuNP size, X-ray properties, and detector geometry, influence the accuracy of Bio-SOSS. This comprehensive investigation highlights the practicality and potential of Bio-SOSS in accurately capturing the diverse conformation spectrum of biological macromolecules, paving the way for deeper insights into their dynamic natures.

show abstract

Visualizing Heterogeneous Protein Conformations with Multi-Tilt Nanoparticle-Aided Cryo-Electron Microscopy Sampling

Cited by 3 publications

References 62 publications

Raman Spectroscopic Insights of Phase-Separated Insulin Aggregates

Raman Spectroscopic Insights of Phase-Separated Insulin Aggregates

Brain Hydrophobic Peptides Antagonists of Neurotoxic Amyloid β Peptide Monomers/Oligomers–Protein Interactions

Single-Molecule X-ray Scattering Used to Visualize the Conformation Distribution of Biological Molecules via Single-Object Scattering Sampling

Contact Info

Product

Resources

About