Quarterly Reviews of Biophysics最新文献

The nucleocapsid protein NC is a crucial component in the human immunodeficiency virus type 1 life cycle. It functions both in its processed mature form and as part of the polyprotein Gag that plays a key role in the formation of new viruses. NC can protect nucleic acids (NAs) from degradation by compacting them to a dense coil. Moreover, through its NA chaperone activity, NC can also promote the most stable conformation of NAs. Here, we explore the balance between these activities for NC and Gag by confining DNA-protein complexes in nanochannels. The chaperone activity is visualized as concatemerization and circularization of long DNA via annealing of short single-stranded DNA overhangs. The first ten amino acids of NC are important for the chaperone activity that is almost completely absent for Gag. Gag condenses DNA more efficiently than mature NC, suggesting that additional residues of Gag are involved. Importantly, this is the first single DNA molecule study of full-length Gag and we reveal important differences to the truncated Δ-p6 Gag that has been used before. In addition, the study also highlights how nanochannels can be used to study reactions on ends of long single DNA molecules, which is not trivial with competing single DNA molecule techniques.

The kink-turn (k-turn) is a widespread structural motif found in functional RNA species. It typically comprises a three-nucleotide bulge followed by tandem trans sugar edge-Hoogsteen G:A base pairs. It introduces a sharp kink into the axis of duplex RNA, juxtaposing the minor grooves. Cross-strand H-bonds form at the interface, accepted by the conserved adenine nucleobases of the G:A basepairs. Alternative acceptors for one of these divides the k-turns into two conformational classes N3 and N1. The base pair that follows the G:A pairs (3b:3n) determines which conformation is adopted by a given k-turn. k-turns often mediate tertiary contacts in folded RNA species and frequently bind proteins. Common k-turn binding proteins include members of the L7Ae family, such as the human 15·5k protein. A recognition helix within these proteins binds in the widened major groove on the outside of the k-turn, that makes specific H-bonds with the conserved guanine nucleobases of the G:A pairs. L7Ae binds with extremely high affinity, and single-molecule data are consistent with folding by conformational selection. The standard, simple k-turn can be elaborated in a variety of ways, that include the complex k-turns and the k-junctions. In free solution in the absence of added metal ions or protein k-turns do not adopt the tightly-kinked conformation. They undergo folding by the binding of proteins, by the formation of tertiary contacts, and some (but not all) will fold on the addition of metal ions. Whether or not folding occurs in the presence of metal ions depends on local sequence, including the 3b:3n position, and the -1b:-1n position (5' to the bulge). In most cases -1b:-1n = C:G, so that the 3b:3n position is critical since it determines both folding properties and conformation. In general, the selection of these sequence matches a given k-turn to its biological requirements. The k-turn structure is now very well understood, to the point at which they can be used as a building block for the formation of RNA nano-objects, including triangles and squares.

Copper is a redox-active transition metal ion required for the function of many essential human proteins. For biosynthesis of proteins coordinating copper, the metal may bind before, during or after folding of the polypeptide. If the metal binds to unfolded or partially folded structures of the protein, such coordination may modulate the folding reaction. The molecular understanding of how copper is incorporated into proteins requires descriptions of chemical, thermodynamic, kinetic and structural parameters involved in the formation of protein-metal complexes. Because free copper ions are toxic, living systems have elaborate copper-transport systems that include particular proteins that facilitate efficient and specific delivery of copper ions to target proteins. Therefore, these pathways become an integral part of copper protein folding in vivo. This review summarizes biophysical-molecular in vitro work assessing the role of copper in folding and stability of copper-binding proteins as well as protein-protein copper exchange reactions between human copper transport proteins. We also describe some recent findings about the participation of copper ions and copper proteins in protein misfolding and aggregation reactions in vitro.

Protein misfolding in cells is avoided by a network of protein chaperones that detect misfolded or partially folded species. When proteins escape these control systems, misfolding may result in protein aggregation and amyloid formation. We here show that aggregation of the amyloidogenic protein α-synuclein (αS), the key player in Parkinson's disease, is controlled by the copper transport protein Atox1 in vitro. Copper ions are not freely available in the cellular environment, but when provided by Atox1, the resulting copper-dependent ternary complex blocks αS aggregation. Because the same inhibition was found for a truncated version of αS, lacking the C-terminal part, it appears that Atox1 interacts with the N-terminal copper site in αS. Metal-dependent chaperoning may be yet another manner in which cells control its proteome.

In this review, we describe how the interplay among science, technology and community interests contributed to the evolution of four structural biology data resources. We present the method by which data deposited by scientists are prepared for worldwide distribution, and argue that data archiving in a trusted repository must be an integral part of any scientific investigation.

Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 - a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution - reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1-3 regions 'sense' nucleic acids, 'regulate' the HNH conformational transition, and ultimately 'lock' the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1-3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.

The large ribosomal subunit has a distinct feature, the stalk, extending outside the ribosome. In bacteria it is called the L12 stalk. The base of the stalk is protein uL10 to which two or three dimers of proteins bL12 bind. In archea and eukarya P1 and P2 proteins constitute the stalk. All these extending proteins, that have a high degree of flexibility due to a hinge between their N- and C-terminal parts, are essential for proper functionalization of some of the translation factors. The role of the stalk proteins has remained enigmatic for decades but is gradually approaching an understanding. In this review we summarise the knowhow about the structure and function of the ribosomal stalk till date starting from the early phase of ribosome research.

RecA family proteins pair two DNAs with the same sequence to promote strand exchange during homologous recombination. To understand how RecA proteins search for and recognize homology, we sought to determine the length of homologous sequence that permits RecA to start its reaction. Specifically, we analyzed the effect of sequence heterogeneity on the association rate of homologous DNA with RecA/single-stranded DNA complex. We assumed that the reaction can start with equal likelihood at any point in the DNA, and that sequence heterogeneity abolishes some possible initiation sites. This analysis revealed that the effective recognition size is five or six nucleotides, larger than the three nucleotides recognized by a RecA monomer. Because the first DNA is elongated 1.5-fold by intercalation of amino acid residues of RecA every three bases, the second bound DNA must be elongated to pair with the first. Because this length is similar to estimates based on the strand-exchange reaction or DNA pair formation, the homology test is likely to occur primarily at the association step. The energetic difference due to the absence of hydrogen bonding is too small to discriminate single-nucleotide heterogeneity over a five- or six-nucleotide sequence. The selection is very likely to be made kinetically, and probably involves some structural factor other than Watson-Crick hydrogen bonding. It would be valuable to determine whether this is also the case for other biological reactions involving DNA base complementarity, such as replication, transcription, and translation.