Role of counterion condensation in folding of the Tetrahymena ribozyme. II. Counterion-dependence of folding kinetics

Condensed counterions contribute to the stability of compact structures in RNA, largely by reducing electrostatic repulsion among phosphate groups. Varieties of cations induce a collapsed state in the Tetrahymena ribozyme that is readily transformed to the catalytically active structure in the presence of Mg2+. Native gel electrophoresis was used to compare the effects of the valence and size of the counterion on the kinetics of this transition. The rate of folding was found to decrease with the charge of the counterion. Transitions in monovalent ions occur 20- to 40-fold faster than transitions induced by multivalent metal ions. These results suggest that multivalent cations yield stable compact structures, which are slower to reorganize to the native conformation than those induced by monovalent ions. The folding kinetics are 12-fold faster in the presence of spermidine3+ than [Co(NH3)6]3+, consistent with less effective stabilization of long-range RNA interactions by polyamines. Under most conditions, the observed folding rate decreases with increasing counterion concentration. In saturating amounts of counterion, folding is accelerated by addition of urea. These observations indicate that reorganization of compact intermediates involves partial unfolding of the RNA. We find that folding of the ribozyme is most efficient in a mixture of monovalent salt and Mg2+. This is attributed to competition among counterions for binding to the RNA. The counterion dependence of the folding kinetics is discussed in terms of the ability of condensed ions to stabilize compact structures in RNA.     

Slow folding kinetics of RNase P RNA.

Understanding the folding mechanisms of large, highly structured RNAs is important for understanding how these molecules carry out their function. Although models for the three-dimensional architecture of several large RNAs have been constructed, the process by which these structures are formed is only now beginning to be explored. The kinetic folding pathway of the Tetrahymena ribozyme involves multiple intermediates and both Mg2+-dependent and Mg2+-independent steps. To determine whether this general mechanism is representative of folding of other large RNAs, a study of RNase P RNA folding was undertaken. We show, using a kinetic oligonucleotide hybridization assay, that there is at least one slow step on the folding pathway of RNase P RNA, resulting in conformational changes in the P7 helix region on the minute timescale. Although this folding event requires the presence of Mg2+, the slow step itself does not involve Mg2+ binding. The P7 and P2 helix regions exhibit distinctly different folding behavior and ion dependence, implying that RNase P folding is likely to be a complex process. Furthermore, there are distinct similarities in the folding of RNase P RNA from both Bacillus subtilis and Escherichia coli, indicating that the folding pathway may also be conserved along with the final structure. The slow folding kinetics, Mg2+-independence of the rate, and existence of intermediates are basic features of the folding mechanism of the Tetrahymena group I intron that are also found in RNase P RNA, suggesting these may be general features of the folding of large RNAs.     

Energy barriers, pathways, and dynamics during folding of large, multidomain RNAs

Large, multidomain RNA molecules are generally thought to fold following multiple pathways down rugged landscapes populated with intermediates and traps. A challenge to understanding RNA folding reactions is the complex relationships that exist between the structure of the RNA and its folding landscape. The identification of intermediate species that populate folding landscapes and characterization of elements of their structures are the key components to solving the RNA folding problem. This review explores recent studies that characterize the dominant pathways by which RNA folds, structural and dynamic features of intermediates that populate the folding landscape, and the energy barriers that separate the distinct steps of the folding process.     

Protein-Facilitated Folding of Group II Intron Ribozymes

Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5γ group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and dimethyl sulfate structural probing, we monitored Mss-116-facilitated folding of ai5γ intron ribozymes and a catalytically active self-splicing RNA containing full-length intron and short exons. We found that the protein directly stimulates folding of these RNAs by accelerating formation of the compact near-native state. This process occurs in an ATP-independent manner, although ATP is required for the protein turnover. As Mss 116 binds RNA nonspecifically, most binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such nonproductive complexes and rebind the unfolded RNA. Results obtained from experiments at different concentrations of magnesium ions suggest that Mss 116 stimulates folding of ai5γ ribozymes by promoting the formation of unstable folding intermediates, which is then followed by a cascade of folding events resulting in the formation of the compact near-native state. Dimethyl sulfate probing results suggest that the compact state formed in the presence of the protein is identical to the near-native state formed more slowly in its absence. Our results also indicate that Mss 116 does not stabilize the native state of the ribozyme, but that such stabilization results from binding of attached exons.     

Reversible folding of cysteine-rich metallothionein by an overcritical reaction path

A first-order-like state transition is considered to be involved in the restoration of the activities of a few proteins by correctly folding the protein [Phys. Rev. E 66 (2002) 021903]. In order to understand the general applicability of this mechanism, we studied a metallothionein (MT) protein with an unconventional structure, i.e., without any alpha-helix or beta-sheet. MT is a 61 amino-acid peptide. There are 6-7 Zn(2+) ions, which bind avidly to 20 conserved cysteines (Cys) of MT. These properties indicate that the structure of MT is quite different from those of the other proteins. Similar to our previous findings, the denatured MT can be folded without any aggregation via a designated stepwise quasi-static process (an over-critical reaction path). The particle size of folded MT intermediates, determined by dynamic light scattering, shrank right after the first folding stage. It is consistent with a collapse-model. In addition, results from both atomic absorption and circular dichroism (CD) indicate that the stable intermediates may fold to the native conformation but with only partial Zn(2+) binding, which in turn implies that those folding intermediates are in a molten globular state. These reversible unfolding and folding processes indicate that Cys-rich protein, MT, may also be folded by way of a first-order-like state transition mechanism. We suspect that this process may likely be involved in the reaction of the metal substitution process in metal containing enzymes.     

Energy barriers, cooperativity, and hidden intermediates in the folding of small proteins

Current theoretical views of the folding process of small proteins (< approximately 100 amino acids) postulate that the landscape of potential mean force (PMF) for the formation of the native state has a funnel shape and that the free energy barrier to folding arises from the chain configurational entropy only. However, recent theoretical studies on the formation of hydrophobic clusters with explicit water suggest that a barrier should exist on the PMF of folding, consistent with the fact that protein folding generally involves a large positive activation enthalpy at room temperature. In addition, high-resolution structural studies of the hidden partially unfolded intermediates have revealed the existence of non-native interactions, suggesting that the correction of the non-native interactions during folding should also lead to barriers on PMF. To explore the effect of a PMF barrier on the folding behavior of proteins, we modified Zwanzig's model for protein folding with an uphill landscape of PMF for the formation of transition states. We found that the modified model for short peptide segments can satisfy the thermodynamic and kinetic criteria for an apparently two-state folding. Since the Levinthal paradox can be solved by a stepwise folding of short peptide segments, a landscape of PMF with a locally uphill search for the transition state and cooperative stabilization of folding intermediates/native state is able to explain the available experimental results for small proteins. We speculate that the existence of cooperative hidden folding intermediates in small proteins could be the consequence of the highly specific structures of the native state, which are selected by evolution to perform specific functions and fold in a biologically meaningful time scale.     

Principles of RNA compaction: insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations

Counterions are required for RNA folding, and divalent metal ions such as Mg(2+) are often critical. To dissect the role of counterions, we have compared global and local folding of wild-type and mutant variants of P4-P6 RNA derived from the Tetrahymena group I ribozyme in monovalent and in divalent metal ions. A remarkably simple picture of the folding thermodynamics emerges. The equilibrium folding pathway in monovalent ions displays two phases. In the first phase, RNA molecules that are initially in an extended conformation enforced by charge-charge repulsion are relaxed by electrostatic screening to a state with increased flexibility but without formation of long-range tertiary contacts. At higher concentrations of monovalent ions, a state that is nearly identical to the native folded state in the presence of Mg(2+) is formed, with tertiary contacts that involve base and backbone interactions but without the subset of interactions that involve specific divalent metal ion-binding sites. The folding model derived from these and previous results provides a robust framework for understanding the equilibrium and kinetic folding of RNA.     

Aromatic and methyl NOEs highlight hydrophobic clustering in the unfolded state of an SH3 domain

The N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) exists in equilibrium between a folded (F(exch)) state and a relatively compact unfolded (U(exch)) state under nondenaturing conditions. Selectively labeled samples of the domain have been analyzed by NOESY NMR experiments to probe residual hydrophobic clustering in the U(exch) state. The labeling strategy included selective protonation of aromatic rings or delta-methyl groups on Ile and Leu residues in a highly deuterated background. Combined with long mixing times, the methods permitted observation of significant numbers of long-range interactions between hydrophobic side chains, providing evidence for multiple conformers involving non-native hydrophobic clusters around the Trp 36 indole. Comparison of these data with previously reported HN-HN NOEs yields structural insight into the diversity of structures within the U(exch) ensemble in the drkN SH3 domain. Many of the HN-HN NOEs are consistent with models containing compact residual nativelike secondary structure and greater exposure of the Trp 36 indole to solvent, similar to kinetic intermediates formed in the hierarchic condensation model of folding. However, the methyl and aromatic NOE data better fit conformations with non-native burial of the Trp indole surrounded by hydrophobic groups and more loosely formed beta-structure; these structural characteristics are more consistent with those of kinetic intermediates formed during the hydrophobic collapse mechanism of folding. This suite of NOE data provides a more complete picture of the structures that span the U(exch) state ensemble, from conformers with non-native structure but long-range contacts to those that are highly nativelike. Together, the results are also consistent with the folding funnel view involving multiple folding pathways for this molecule.     

RNA tertiary interactions mediate native collapse of a bacterial group I ribozyme

Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.     

Atomistic Picture for the Folding Pathway of a Hybrid-1 Type Human Telomeric DNA G-quadruplex

In this work we studied the folding process of the hybrid-1 type human telomeric DNA G-quadruplex with solvent and ions explicitly modeled. Enabled by the powerful bias-exchange metadynamics and large-scale conventional molecular dynamic simulations, the free energy landscape of this G-DNA was obtained for the first time and four folding intermediates were identified, including a triplex and a basically formed quadruplex. The simulations also provided atomistic pictures for the structures and cation binding patterns of the intermediates. The results showed that the structure formation and cation binding are cooperative and mutually supporting each other. The syn/anti reorientation dynamics of the intermediates was also investigated. It was found that the nucleotides usually take correct syn/anti configurations when they form native and stable hydrogen bonds with the others, while fluctuating between two configurations when they do not. Misfolded intermediates with wrong syn/anti configurations were observed in the early intermediates but not in the later ones. Based on the simulations, we also discussed the roles of the non-native interactions. Besides, the formation process of the parallel conformation in the first two G-repeats and the associated reversal loop were studied. Based on the above results, we proposed a folding pathway for the hybrid-1 type G-quadruplex with atomistic details, which is new and more complete compared with previous ones. The knowledge gained for this type of G-DNA may provide a general insight for the folding of the other G-quadruplexes.     

Modeling the role of disulfide bonds in protein folding: Entropic barriers and pathways

The role of disulfide bonds in directing protein folding is studied using lattice models. We find that the stability and the specificity of the disulfide bond interactions play quite different roles in the folding process: Under some conditions, the stability decreases the overall rate of folding; the specificity, however, by yielding a simpler connectivity of intermediates, always increases the rate of folding. This conclusion is intimately related to the selection mechanism entailed by entropic driving forces, such as the loop formation probability, and entropic barriers separating the native and the many native-like metastable states. The folding time is found to be a minimum for a certain range of the effective disulfide bond interaction. Examination of a model, which allows for the formation of disulfide bonded intermediates, suggests that folding proceeds via a threestage multiple pathways kinetics. We show that there are pathways to the native state involving only native-like intermediates, as well as those that are mediated by nonnative intermediates. These findings are interpreted in terms of the appropriate energy landscape describing the barriers connecting low energy conformations. The consistency of our conclusions with several experimental studies is also discussed. © 1995 Wiley-Liss, Inc.     

Beta-hairpin folding simulations in atomistic detail using an implicit solvent model

We have used distributed computing techniques and a supercluster of thousands of computer processors to study folding of the C-terminal beta-hairpin from protein G in atomistic detail using the GB/SA implicit solvent model at 300 K. We have simulated a total of nearly 38 micros of folding time and obtained eight complete and independent folding trajectories. Starting from an extended state, we observe relaxation to an unfolded state characterized by non-specific, temporary hydrogen bonding. This is followed by the appearance of interactions between hydrophobic residues that stabilize a bent intermediate. Final formation of the complete hydrophobic core occurs cooperatively at the same time that the final hydrogen bonding pattern appears. The folded hairpin structures we observe all contain a closely packed hydrophobic core and proper beta-sheet backbone dihedral angles, but they differ in backbone hydrogen bonding pattern. We show that this is consistent with the existing experimental data on the hairpin alone in solution. Our analysis also reveals short-lived semi-helical intermediates which define a thermodynamic trap. Our results are consistent with a three-state mechanism with a single rate-limiting step in which a varying final hydrogen bond pattern is apparent, and semi-helical off-pathway intermediates may appear early in the folding process. We include details of the ensemble dynamics methodology and a discussion of our achievements using this new computational device for studying dynamics at the atomic level.     

Topological and energetic factors: what determines the structural details of the transition state ensemble and "en-route" intermediates for protein folding? An investigation for small globular proteins

Recent experimental results suggest that the native fold, or topology, plays a primary role in determining the structure of the transition state ensemble, at least for small, fast-folding proteins. To investigate the extent of the topological control of the folding process, we studied the folding of simplified models of five small globular proteins constructed using a Go-like potential to retain the information about the native structures but drastically reduce the energetic frustration and energetic heterogeneity among residue-residue native interactions. By comparing the structure of the transition state ensemble (experimentally determined by Phi-values) and of the intermediates with those obtained using our models, we show that these energetically unfrustrated models can reproduce the global experimentally known features of the transition state ensembles and "en-route" intermediates, at least for the analyzed proteins. This result clearly indicates that, as long as the protein sequence is sufficiently minimally frustrated, topology plays a central role in determining the folding mechanism.     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

Role of metal ions in the tetraloop-receptor complex as analyzed by NMR

Metal ions are critical for the proper folding of RNA, and the GAAA tetraloop-receptor is necessary for the optimal folding and function of many RNAs. We have used NMR to investigate the role of metal ions in the structure of the tetraloop-receptor in solution. The NMR data indicate native tertiary structure is formed under a wide range of ionic conditions. The lack of conformational adaptation in response to very different ionic conditions argues against a structural role for divalent ions. Nuclear Overhauser effects to cobalt hexammine and paramagnetic relaxation enhancement induced by manganese ions were used to determine the NMR structures of the tetraloop receptor in association with metal ions, providing the first atomic-level view of these interactions in the solution state. Five manganese and two cobalt hexammine ions could be localized to the RNA surface. The locations of the associated metal ions are similar, but not identical to, those of previously determined crystal structures. The sites of association are in general agreement with nonlinear Poisson-Boltzmann calculations of the electrostatic surface, emphasizing the general importance of diffusely associated ions in RNA tertiary structure.     

GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism.

The chaperonin GroEL binds folding intermediates of four-disulfidehen lysozyme transiently within its central cavity. Using stopped flow fluorescence we show that GroEL binds early intermediates in folding and accelerates the slow kinetic phase that reflects the reversal of non-native interactions involving tryptophan residues and the formation of the native state. Pulsed hydrogen exchange monitored by electrospray ionization mass spectrometry demonstrates that GroEL does not alter the folding mechanism, nor are protected species unfolded by the chaperonin. The data suggest a mechanism for GroEL-assisted folding in which the reorganization of non-native tertiary interactions is facilitated but domain folding is unperturbed.