No abstract

We investigate the microsecond-timescale kinetics of the RNA hairpin ga*cUUCGguc. The fluorescent nucleotide 2-aminopurine (a*) reports mainly on base stacking. Ten kinetic traces and the temperature denaturation curve are globally fitted to four-state models of the free-energy surface. In the best-fitting sequential model, the hairpin unfolds over successively larger barriers in at least three stages: stem fraying and increased base-stacking fluctuations; concerted loss of hydrogen bonding and partial unstacking; and additional unstacking of single strands at the highest temperatures. Parallel and trap models also provide adequate fits: such pathways probably also play a role in the complete free-energy surface of the hairpin. To interpret the model states structurally, 200 ns of molecular dynamics, including six temperature-jump simulations, were run. Although the sampling is by no means comprehensive, five different states were identified using hydrogen bonding and base stacking as reaction coordinates. The four to five states required to explain the experiments or simulations set a lower limit on the complexity of this small RNA hairpin's energy landscape.

2-Aminopurine (2AP) is a fluorescent adenine analog that probes mainly base stacking in nucleic acids. We labeled the loop or the stem of the RNA hairpin gacUACGguc with 2AP to study folding thermodynamics and kinetics at both loci. Thermal melts and fast laser temperature jumps detected by 2AP fluorescence monitored the stability and folding/unfolding kinetics. The observed thermodynamic and kinetic traces of the stem and loop mutants, though strikingly different at a first glance, can be fitted to the same free-energy landscape. The differences between the two probe locations arise because base stacking decreases upon unfolding in the stem, whereas it increases in the loop. We conclude that 2AP is a conservative adenine substitution for mapping out the contributions of different RNA structural elements to the overall folding process. Molecular dynamics (MD) totaling 0.6 msec were performed to look at the conformations populated by the RNA at different temperatures. The combined experimental data, and MD simulations lead us to propose a minimal four-state free-energy landscape for the RNA hairpin. Analysis of this landscape shows that a sequential folding model is a good approximation for the full folding dynamics. The frayed state formed initially from the native state is a heterogeneous ensemble of structures whose stem is frayed either from the end or from the loop.

Although structure and sequence signatures in ribosomal RNA and proteins are defining characteristics of the three domains of life and instrumental in constructing the modern phylogeny, little is known about their functional roles in the ribosome. In this work, the largest coevolving RNA/protein signatures in the bacterial 30S ribosome are investigated both experimentally and computationally through all-atom molecular-dynamics simulations. The complex includes the N-terminal fragment of the ribosomal protein S4, which is a primary binding protein that initiates 30S small subunit assembly from the 5' domain, and helix 16 (h16), which is part of the five-way junction in 16S rRNA. Our results show that the S4 N-terminus signature is intrinsically disordered in solution, whereas h16 is relatively stable by itself. The dynamic disordered property of the protein is exploited to couple the folding and binding process to the five-way junction, and the results provide insight into the mechanism for the early and fast binding of S4 in the assembly of the ribosomal small subunit.

Lambda repressor fragment λ(*)(6-85) is one of the fastest folding small protein fragments known to date. We hypothesized that removal of three out of five helices of λ(*)(6-85) would further reduce this protein to its smallest folding core. Molecular dynamics simulations singled out two energetically stable reduced structures consisting of only helices 1 and 4 connected by a short glycine/serine linker, as well as a less stable control. We investigated these three polypeptides and their fragments experimentally by using circular dichroism, fluorescence spectroscopy, and temperature jump relaxation spectroscopy to gain insight into their thermodynamic and kinetic properties. Based on the thermal melts, the order of peptide stability was in correspondence with theoretical predictions. The most stable two-helix bundle, λ(blue1), is a cooperatively folding miniprotein with the same melting temperature and folding rate as the full-length λ(*)(6-85) pseudo wild type and a well-defined computed structure.