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Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size‐distribution profile of samples containing microscopic particles in suspension or solubilized polymers. In this work, Raynals, user‐friendly software for the analysis of single‐angle DLS data that uses the Tikhonov–Phillips regularization, is introduced. Its performance is evaluated on simulated and experimental data generated by different DLS instruments for several proteins and gold nanoparticles. DLS data can easily be misinterpreted and the simulation tools available in Raynals allow the limitations of the measurement and its resolution to be understood. It was designed as a tool to address the quality control of biological samples during sample preparation and optimization and it helps in the detection of aggregates, showing the influence of large particles. Lastly, Raynals provides flexibility in the way that the data are presented, allows the export of publication‐quality figures, is free for academic use and can be accessed online on the eSPC data‐analysis platform at https://spc.embl‐hamburg.de/. [ABSTRACT FROM AUTHOR]

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision (1). The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase--[tRNA.sub.CUA] (MjTyrRS-[tRNA.sub.CUA]) (2,3) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase--[tRNA.sub.CUA] (MbPylRS-[tRNA.sub.CUA]) (4-6) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli (1,6,7). However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (8,9) (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates (8).By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids (10). Because the synthetase--tRNA pairs used have been evolved to incorporate numerous unnatural amino acids (1,6,7), it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells., Because each of the 64 triplet codons are used to encode natural amino acids or polypeptide termination, new blank codons are required for cellular genetic code expansion. In principle, quadruplet [...]

An essential element of adaptive immunity is selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes. Using native mass spectrometry, we analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule and, due to its unique stability, we determine binding affinities of complexes loaded with truncated or charge-reduced peptides. We find that the two anchor positions can be stabilized independently, and we further analyze the contribution of additional amino acid positions to the binding strength. As a complement to computational prediction tools, our method estimates binding strength of even low-affinity peptides to MHC class I complexes quickly and efficiently. It has huge potential to eliminate binding affinity biases and thus accelerate drug discovery in infectious diseases, autoimmunity, vaccine design, and cancer immunotherapy. The authors present a sensitive and rapid method to determine the binding strength of MHC class 1 peptide complexes using native mass spectrometry. [ABSTRACT FROM AUTHOR]

Research has been conducted on the human papillomavirus E7 oncoprotein. The isolation and characterization of E7 dimer have been carried out and the results indicate that this dimer undergoes structural transitions which generate hydrophobic surfaces accessible to solvent upon pH changes.

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm, should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X‐ray crystallography and cryo‐electron microscopy, screening for small‐molecule binding partners and comparison of effects of point mutations. [ABSTRACT FROM AUTHOR]

The interaction of the human papillomavirus E7 oncoprotein (HPV16 E7) with the DNA binding domain of the E2 protein (E2C) which leads to ionic strength-dependent hetero-oligomerization even at the lowest measurable concentrations is described. Sequestering E2 into E7-E2 oligomers is found to provide a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome.

A study was conducted to show that recombinant high-risk HPV 16 and HPV 18 E6 readily fold into very stable soluble oligomers (E6SOs) that exhibit secondary and tertiary structure, cooperatively lost upon denaturation. The results indicate that the cytoplasmic species contain conformational epitopes that might arise from yet undefined homo or hetero-oligomers, but its localization otherwise agrees with that of the other group of major E6 targets.

The E7 oncoprotein is the major transforming activity in human papillomavirus and shares sequence and functional properties with adenovirus E1A and SV40 T-antigen by targeting the pRb tumor suppressor. The N-terminal domain of E7 can interfere with pRb binding but not with the chaperone activity, which requires the C-terminal domain, in most of the E7 activities.

The ability of HPV16 E7, a major transforming oncoprotein in papillomavirus, to self-assemble in the absence of zinc into defined spherical oligomer is explained. The conformational characterization of these structures is done by means of electron microscopy, dynamic light scattering and spectroscopic techniques like circular dichroism and fluorescence.

Human syncytial respiratory virus is a nonsegmented negative‐strand RNA virus with serious implications for respiratory disease in infants, and has recently been reclassified into a new family, Pneumoviridae. One of the main reasons for this classification is the unique presence of a transcriptional antiterminator, called M2–1. The puzzling mechanism of action of M2–1, which is a rarity among antiterminators in viruses and is part of the RNA polymerase complex, relies on dissecting the structure and function of this multidomain tetramer. The RNA‐binding activity is located in a monomeric globular `core' domain, a high‐resolution crystal structure of which is now presented. The structure reveals a compact domain which is superimposable on the full‐length M2–1 tetramer, with additional electron density for the C‐terminal tail that was not observed in the previous models. Moreover, its folding stability was determined through chemical denaturation, which shows that the secondary and tertiary structure unfold concomitantly, which is indicative of a two‐state equilibrium. These results constitute a further step in the understanding of this unique RNA‐binding domain, for which there is no sequence or structural counterpart outside this virus family, in addition to its implications in transcription regulation and its likeliness as an antiviral target. [ABSTRACT FROM AUTHOR]