Formation Of Tertiary Interactions Research Articles

Formation of Tertiary Interactions during rRNA GTPase Center Folding

The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg2+ ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg2+-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg2+ addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring from 5ms to 4s and with global folding from 1 to 5s. Site-specific substitution with 15N-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg2+ and suggest that there are two initial sites of Mg2+ ion association.

Denaturant Probes Quantify the Preferential Burial of Amide Surface in Forming the Key Transition State and Early Intermediates in Protein Folding

Protein-folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early-unstable-intermediates and the high-free-energy transition-state (TS) will help answer some of these. Recently we used effects of denaturants (urea, GuHCl) and temperature on folding and unfolding rate constants and overall-equilibrium constant, as probes of surface-area changes in protein-folding. We interpreted denaturant kinetic m-values and activation heat-capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding (Guinn et al, PNAS '13). Predicted accessible-surface-area (ASA) changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50-90% of overall surface-burial) and that surface buried is disproportionately amide, demonstrating extensive formation of secondary-structure in early-intermediates. Models of possible pre-TS intermediates with all elements of the native secondary-structure for several of these proteins, created and analyzed using PyMol, Coot, and Surfracer, bury less amide and hydrocarbon surface than predicted for TS. Extensions of this approach to deduce models for transition-states and early-intermediates in folding of other proteins in the data set are in progress to test the proposal that TS generally has both native secondary-structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. To facilitate these analyses, we have developed a program (Calculate_Unfolded_ASA.py) to predict ASA of fully-extended unfolded state, shown to be a good model for denaturant-induced unfolding. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.

The Challenge to the Rule of Homology Modeling: Folding Mechanism Study of Protein GA and GB with High Sequence Identity but Different Native Structures

As one of the most valuable methods for drug design, homology modeling shows that protein structures are more conserved than protein sequences, that is, the proteins with high sequence identity have high structural similarity, but protein pairs G(A)88/G(B)88 and G(A)95/G(B)95 prove the opposite. The pairs G(A)88 and G(B)88 shares the 88% sequence identity, but display different structures, and the pair G(A)95 and G(B)95 with 95% sequence identity yet presents different structures. The research on these proteins provides an opportunity of complementary study. In the process of protein folding, at which stage the protein final structure was determined and which residues were important for folding to a given structure were still unknown. Here we used OPLS all-atom force field for molecular dynamics simulations to study the unfolding of G(A)88, G(B)88, G(A)95 and G(B)95 at high temperatures, and used the process of protein unfolding to reverse the process of protein folding. G(B)88 and G(B)95 folded to the α+β structure, but G(A)88 and G(A)95 folded to the all-α-helix structure. In the process of G(A)88 and G(A)95 folding, the helices folded earlier than the formation of tertiary interactions. In the process of folding to G(B)88 and G(B)95, the α-helix formed earlier. We showed that early along the folding pathway, the final protein structure was confirmed, and very small differences between protein sequences determined the protein structure.

The structure and folding of kink turns in RNA

The kink turn (k-turn) is a widespread structural motif that introduces a tight kink into the axis of double-stranded RNA, with an included angle ∼60°. A standard k-turn comprises a three-nucleotide bulge followed on the 3' side by a G•A pair, an A•G pair, and usually further non-Watson-Crick pairs. The kinked conformation may be stabilized by three processes. These are the addition of metal ions, the binding of proteins such as the L7Ae family, and by the formation of tertiary interactions. The structure is characterized by specific A-minor interactions with the adenine nucleobases of the G•A pairs, and some very well-conserved hydrogen bonds involving 2'-hydroxyl groups. We can identify two classes of k-turns, that differ in the manner of the hydrogen bonding at the adenine of the bulge-distal G•A pair.

Unfolding Single RNA Molecules with Optical Tweezers

RNA molecules must fold into specific three-dimensional shapes to perform their structural and catalytic functions. Unlike proteins, RNAs secondary structural features are usually stable enough to form by themselves in solution. The reason is that in RNA, the stabilization energy gained from the formation of secondary structure is substantially larger than the energies involved in tertiary interactions. As a result, the formation of tertiary interactions is expected to alter only slightly the pre-existing secondary structural contacts. Moreover, secondary structure prediction is robust and can be made without taking into consideration tertiary folding. However, bulk studies of the energetics and kinetics of their secondary and tertiary folding are often frustrated by the presence of multiple species and multiple folding pathways in solution. These problems are circumvented in single-molecule studies in which the folding/unfolding trajectories of the individual molecules can be followed. The T. thermophila group I intron ribozyme is organized into several domains whose mechanical unfolding can be investigated independently, and whose tertiary contacts are stabilized by numerous Mg++ ions.We have begun characterization of the ribozyme by analysis of the P5abc domain because: