Minimum energy conformations of DNA dimeric subunits: Potential energy calculations for dGpdC, dApdA, dCpdC, dGpdG, and dTpdT

Minimum energy conformations have been calculated for the deoxydinucleoside phosphates dGpdC, dApdA, dCpdC, dGpdG, and dTpdT. In these potential energy calculations the eight diheldral angles and the sugar pucker were flexible parameters. A substantial survey of conformation space was made in which all staggred combination of the dihedral angles ω′,ω, and ψ, in conjunction with C(2′)-endo pucker, were used as starting conformers for the energy minimization. The most important conformations in the C(3′)-endo-puckering domain have ψ = g+; ω′,ω = g−,g−(A-form),g+, g+, and g−,t. With C(2′)-endo-type pucker the most important conformations have ψ = g+; ω′,ω =g-,g-(B-form) and g+,t; and ψ =t; ω′,ω =g-,t(Watson-Crick from) and t,g+ (skewed). Stacked bases are a persistent feature of the low-energy conformations, the g+ conformer being an exception. Freeing the suger pucker allowed this conformation to become low energy, with C(3′)-exo pucker. It also caused other low-energy forms, such and the Waston-Crick conformation, to become more favourable. Conformation flexibility in the sugar pucker and in ψ, as well as the ω′,ω angle pair, is indicated for the dimeric subunits of DNA

Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors.

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer

Folding and Assembly of Multimeric Proteins: Dimeric HIV-1 Protease and a Trimeric Coiled Coil Component of a Complex Hemoglobin Scaffold: A Dissertation

Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function

Snapshots of a molecular swivel in action

Members of the serine family of site-specific recombinases exchange DNA strands via 180° rotation about a central protein-protein interface. Modeling of this process has been hampered by the lack of structures in more than one rotational state for any individual serine recombinase. Here we report crystal structures of the catalytic domains of four constitutively active mutants of the serine recombinase Sin, providing snapshots of rotational states not previously visualized for Sin, including two seen in the same crystal. Normal mode analysis predicted that each tetramer's lowest frequency mode (i.e. most accessible large-scale motion) mimics rotation: two protomers rotate as a pair with respect to the other two. Our analyses also suggest that rotation is not a rigid body movement around a single symmetry axis but instead uses multiple pivot points and entails internal motions within each subunit

The role of the conformational plasticity in the DNA-protein recognition mechanism: NMR study of the Human Papillomavirus E2 system

Maurizio Delfini, Bruno Samorì, Roberto Fttoruss

3D structure of Thermus aquaticus single-stranded DNA–binding protein gives insight into the functioning of SSB proteins

In contrast to the majority of tetrameric SSB proteins, the recently discovered SSB proteins from the Thermus/Deinoccus group form dimers. We solved the crystal structures of the SSB protein from Thermus aquaticus (TaqSSB) and a deletion mutant of the protein and show the structure of their ssDNA binding domains to be similar to the structure of tetrameric SSBs. Two conformations accompanied by proline cis–trans isomerization are observed in the flexible C-terminal region. For the first time, we were able to trace 6 out of 10 amino acids at the C-terminus of an SSB protein. This highly conserved region is essential for interaction with other proteins and we show it to adopt an extended conformation devoid of secondary structure. A model for binding this region to the χ subunit of DNA polymerase III is proposed. It explains at a molecular level the reason for the ssb113 phenotype observed in Escherichia coli

Reconstitution of bacterial cytokinesis: the Z-ring

Prokaryotic cell division is one of the most fundamental processes in biology, but the dynamics and mechanics are far from being understood. In many bacteria, FtsZ, a tubulin homologue assembles into a ring-like structure – Z-ring at precisely the middle of the cell. This accurate site selection is dependent on the Min proteins. Min D and MinE self-organise into waves in vitro, and oscillate pole to pole in vivo. MinC is thought to couple the Min oscillations to FtsZ by direct interaction. The mechanism of inhibitory action of MinC on FtsZ assembly is not known. Critical to the understanding of regulation of FtsZ by MinC and other proteins and its probable role in force generation is the organisation, structure and the dynamics of the Z-ring. Current models of the FtsZ filament organization in the Z-ring argue between two different structures – (i) short overlapping protofilaments with lateral interactions and (ii) few long annealed protofilaments with or without lateral contacts. Our observations of the characteristics of polymerization and turnover studies using fluorescence microscopy suggest that the FtsZ filament is a continuous and irresolute bundle. The results are consistent with a structure where the turnover happens throughout, and any specialised structure resulting in a GTP cap like structure can be ruled out. We show that the turnover rates and hydrolysis rates are similar arguing for a model in which subunit leaves as soon as it hydrolyses GTP. On the basis of crystal structures, we cloned the N-terminal of FtsZ, which acts as a C-terminal end capping fragment and is able to interact with monomers. The end-capping fragment, NZ can disassemble the FtsZ polymers, without influencing the GTPase activity, offering a comparable standard for the activity of MinC. On the basis of our observations, we propose a model on how MinC can disassemble FtsZ polymers. Furthermore, our data shows that the Min CDE system is sufficient to cause spatial regulation of FtsZ provided FtsZ is dynamic. How the Z-ring takes the form of a functional helical or ring-like structure remains unclear. Extensive modelling approaches have tried to explain the ring formation and force generation. Previous studies have qualitatively shown bending of liposome membranes by FtsZ filaments. We hypothesised that the presumably intrinsically curved filaments should respond to pre-curved substrates, and the alignment should be quantifiable. This should ascertain whether or not FtsZ has intrinsic curvature and/or actively induces any force. Thus, we investigated how FtsZ filaments respond to a range of curvatures, which mimic different stages of the division process. Our results show that the FtsZ filaments possess intrinsic curvatures as well as spontaneous twist. This facilitates the formation of Z-ring by utilizing geometrical cues. Our results are in agreement with consistent helical FtsZ polymers observed in vivo by Cryo-EM or super resolution microscopy. The alignment of filaments over a range of curvature suggests that the filaments have considerable flexibility, which strongly suggests reconsidering possible mechanisms of force generation. Moreover, the developed assay constitutes a valuable platform to further study proteins involved in modifying curvature of FtsZ filaments. In summary, by reconstituting the FtsZ filament in vitro, we have elucidated the nature of FtsZ filaments. The dynamics of FtsZ filaments allows them to be inhibited by MinC, thus cooperating with the Min waves. The presence of intrinsic curvature and twist facilitates their formation into a ring necessary for the cell to carry out cytokinesis

Characterization of Nucleic Acids and Nucleic Acid Binding Proteins Using NMR

Biological systems are governed by molecular interactions. At the core of all biological systems is the processing of nucleic acids, and preserving the fidelity of the genome is crucial to those systems. This manuscript follows different biological systems interrogated by nuclear magnetic resonance: the structural impact of single ribonucleotide damage in DNA, the formation of oligomeric protein-DNA complexes of an ETS family transcription factor, how methylation affects the charge state of the amino acid arginine and how the base pair opening rates of an oligonucleotide is affected by inosine