Detection of proteins with ascorbic acid-capped gold nanoparticles: a simple and highly sensitive colorimetric assay.

We report a simple and highly sensitive colorimetric method for the detection and quantification of proteins, based on the aggregation of ascorbic acid (AA) capped gold nanoparticles (AuNPs) by proteins. The interactions between our AuNPs and nine different proteins of various sizes and shapes (cytochrome C (12 kDa), lysozyme (14.3 kDa), myoglobin (17 kDa), human serum albumin (66 kDa), bovine serum albumin (66.4 kDa), human transferrin (80 kDa), aldolase (160 kDa), catalase (240 kDa), and human H-ferritin (500 kDa)) generated similar AuNPs-protein absorption spectra in a concentration-dependent manner in the range of 1-15 nM. Upon the addition of a protein, the UV-visible spectra of AuNPs-protein conjugates shifted from 524 nm for the AuNps alone to longer wavelength (600-750 nm) due to the presence of one of these proteins. This bathochromic shift is accompanied by a color change from a cherry red, to dark purple, and then light grey or colorless if excess protein has been added, indicating the formation of AuNPs-protein conjugates followed by protein-induced aggregation of the AuNPs. High-resolution transmission electron microscopy images revealed uniformly distributed spherical nanoparticles with an average size of 27.5 ± 15.2 nm, increasing in size to 39.6 ± 12.9 nm upon the addition of a protein, indicating the formation of AuNPs-protein conjugates in solution. A general mechanism for the protein-induced aggregation of our AuNPs is proposed. The consistent behavior observed with the nine proteins tested in our study suggests that our assay can be universally applied for the quantification of pure proteins in a solution, regardless of size, shape, or molecular weight.

Unveiling the stochastic nature of human heteropolymer ferritin self-assembly mechanism.

Despite ferritin's critical role in regulating cellular and systemic iron levels, our understanding of the structure and assembly mechanism of isoferritins, discovered over eight decades ago, remains limited. Unveiling how the composition and molecular architecture of hetero-oligomeric ferritins confer distinct functionality to isoferritins is essential to understanding how the structural intricacies of H and L subunits influence their interactions with cellular machinery. In this study, ferritin heteropolymers with specific H to L subunit ratios were synthesized using a uniquely engineered plasmid design, followed by high-resolution cryo-electron microscopy analysis and deep learning-based amino acid modeling. Our structural examination revealed unique architectural features during the self-assembly mechanism of heteropolymer ferritins and demonstrated a significant preference for H-L heterodimer formation over H-H or L-L homodimers. Unexpectedly, while dimers seem essential building blocks in the protein self-assembly process, the overall mechanism of ferritin self-assembly is observed to proceed randomly through diverse pathways. The physiological significance of these findings is discussed including how ferritin microheterogeneity could represent a tissue-specific adaptation process that imparts distinctive tissue-specific functions to isoferritins.

Structural basis for the intracellular regulation of ferritin degradation.

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.