Rapid collapse and slow structural reorganisation during the refolding of bovine alpha-lactalbumin.

The refolding of bovine alpha-lactalbumin (BLA) from its chemically denatured state in 6 M GuHCl has been investigated by a variety of complementary biophysical approaches. CD experiments indicate that the species formed in the early stages of refolding of the apo-protein have at least 85 % of the alpha-helical content of the native state, and kinetic NMR experiments show that they possess near-native compactness. Hydrogen exchange measurements using mass spectrometry and NMR indicate that persistent structure in these transient species is located predominantly in the alpha-domain of the native protein and is similar to that present in the partially folded A-state formed by the protein at low pH. The extent of the exchange protection is, however, small, and there is no evidence for the existence of well-defined discrete kinetic intermediates of the type populated in the refolding of the structurally homologous c-type lysozymes. Rather, both mass spectrometric and NMR data indicate that the rate-determining transition from the compact partially structured (molten globule) species to the native state is highly cooperative. The data show that folding in the presence of Ca2+ is similar to that in its absence, although the rate is increased by more than two orders of magnitude. Sequential mixing experiments monitored by fluorescence spectroscopy indicate that this slower folding is not the result of the accumulation of kinetically trapped species. Rather, the data are consistent with a model in which binding of Ca2+ stabilizes native-like contacts in the partially folded species and reduces the barriers for the conversion of the protein to its native state. Taken together the results indicate that folding of BLA, in the presence of its four disulphide bonds, corresponds to one of the limiting cases of protein folding in which rapid collapse to a globule with a native-like fold is followed by a search for native-like side-chain contacts that enable efficient conversion to the close packed native structure.     

Fast and Slow Tracks in Lysozyme Folding Elucidated by the Technique of Disulfide Scrambling

GdmCl (6 M) unfolded lysozyme was previously shown to refold via kinetically partitioned pathways (Kiefhaber in Proc Natl Acad Sci 92:9029–9033, 1995). About 80% of the unfolded lysozyme molecules refold on a slow pathway with well-populated intermediates. The remaining 20% of denatured lysozyme refold on a fast track without detectable intermediate. This kinetic heterogeneity has been proposed to originate from the collapsed state of lysozyme folding. Using the method of disulfide scrambling, we demonstrate in this report that these two populations of unfolded lysozyme can be isolated and analyzed separately. GdmCl (6 M) denatured lysozyme actually comprises two major populations of unfolded isomers, namely X-LYZ-a and X-LYZ-b with molar ratio of about 80:20. X-LYZ-a and X-LYZ-b exist in equilibrium in the unfolded state. Their disulfide structures and CD properties indicate that X-LYZ-a is more extensively unfolded than X-LYZ-b. Refolding experiments using the method of disulfide scrambling also show that folding kinetics of X-LYZ-a is about 8–10 times slower than that of X-LYZ-b and folding intermediates of X-LYZ-a is far more heterogeneous than that of X-LYZ-b. The results highlight the implication of the conformational heterogeneity of 6 M GdmCl denatured proteins for the interpretation of the initial stage of protein folding mechanism.     

Thermodynamics and Kinetics of Single-Chain Monellin Folding with Structural Insights into Specific Collapse in the Denatured State Ensemble

Proteins, which behave as random coils in high denaturant concentrations undergo collapse transition similar to polymers on denaturant dilution. We study collapse in the denatured ensemble of single-chain monellin (MNEI) using a coarse-grained protein model and molecular dynamics simulations. The model is validated by quantitatively comparing the computed guanidinium chloride and pH-dependent thermodynamic properties of MNEI folding with the experiments. The computed properties such as the fraction of the protein in the folded state and radius of gyration (R_g) as function of [GuHCl] are in good agreement with the experiments. The folded state of MNEI is destabilized with an increase in pH due to the deprotonation of the residues Glu24 and Cys42. On decreasing [GuHCl], the protein in the unfolded ensemble showed specific compaction. The R_g of the protein decreased steadily with [GuHCl] dilution due to increase in the number of native contacts in all the secondary structural elements present in the protein. MNEI folding kinetics is complex with multiple folding pathways and transiently stable intermediates are populated in these pathways. In strong stabilizing conditions, the protein in the unfolded ensemble showed transition to a more compact unfolded state where R_g decreased by ≈ 17% due to the formation of specific native contacts in the protein. The intermediate populated in the dominant MNEI folding pathway satisfies the structural features of the dry molten globule inferred from experiments.     

Formation of a molten globule like state in bovine serum albumin at alkaline pH

Little work has been done to understand the folding profiles of multi-domain proteins at alkaline conditions. We have found the formation of a molten globule-like state in bovine serum albumin at pH 11.2 with the help of spectroscopic techniques; like far and near ultra-violet circular dichroism, intrinsic and extrinsic fluorescence spectroscopy. Interestingly, this state has features similar to the acid-denatured state of human serum albumin at pH 2.0 reported by Muzammil et al. (Eur J Biochem 266:26–32, 1999). This state has also shown significant increase in 8-anilino-1-naphthalene-sulfonate (ANS) binding in compare to the native state. At pH 13.0, the protein seems to acquire a state very close to 6 M guanidinium hydrochloride (GuHCl) denatured one. But, reversibility study shows it can regain nearly 40% of its native secondary structure. On the contrary, tertiary contacts have disrupted irreversibly. It seems, withdrawal of electrostatic repulsion leave room for local interactions, but disrupted tertiary contacts fail to regain their original states.     

Multiple Unfolding Events during Native Folding of the Tetrahymena Group I Ribozyme

Despite the ubiquitous nature of misfolded intermediates in RNA folding, little is known about their physical properties or the folding transitions that allow them to continue folding productively. Folding of the Tetrahymena group I ribozyme includes sequential accumulation of two intermediates, termed I_trap and misfolded (M). Here, we probe the structure and folding transition of I_trap and compare them to those of M. Hydroxyl radical and dimethyl sulfate footprinting show that both I_trap and M are extensively structured and crudely resemble the native RNA. However, regions of the core P3-P8 domain are more exposed to solvent in I_trap than in M. I_trap rearranges to continue folding nearly 1000-fold faster than M, and urea accelerates folding of I_trap much less than M. Thus, the rate-limiting transition from I_trap requires a smaller increase in exposed surface. Mutations that disrupt peripheral tertiary contacts give large and nearly uniform increases in re-folding of M, whereas the same mutations give at most modest increases in folding from I_trap. Intriguingly, mutations within the peripheral element P5abc give 5- to 10-fold accelerations in escape from I_trap, whereas ablation of P13, which lies on the opposite surface in the native structure, near the P3-P8 domain, has no effect. Thus, the unfolding required from I_trap appears to be local, whereas the unfolding of M appears to be global. Further, the modest effects from several mutations suggest that there are multiple pathways for escape from I_trap and that escape is aided by loosening nearby native structural constraints, presumably to facilitate local movements of nucleotides or segments that have not formed native contacts. Overall, these and prior results suggest a model in which the global architecture and peripheral interactions of the RNA are achieved relatively early in folding. Multiple folding and re-folding events occur on the predominant pathway to the native state, with increasing native core interactions and cooperativity as folding progresses.     

Refolding and Polymerization Pathways of Neuroserpin

Neuroserpin is a member of the serpin superfamily, and its mutants are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. It has been proposed that neuroserpin polymers are formed by a conformational change in the folded protein. However, an alternative model whereby polymers are formed during protein folding rather than from the folded protein has recently been proposed. We investigated the refolding and polymerization pathways of wild-type neuroserpin (WT) and of the pathogenic mutants S49P and H338R. Upon refolding, denatured WT immediately formed an initial refolding intermediate I_IN and then underwent further refolding to the native form through a late refolding intermediate, I_R. The late-onset mutant S49P was also able to refold to the native form through I_IN and I_R, but the final refolding step proceeded at a slower rate and with a lower refolding yield as compared with WT. The early-onset mutant H338R formed I_R through the same pathway as S49P, but the protein could not attain the native state and remained as I_R. The I_Rs of the mutants had a long lifespan at 4 °C and thus were purified and characterized. Strikingly, when incubated under physiological conditions, I_R formed ordered polymers with essentially the same properties as the polymers formed from the native protein. The results show that the mutants have a greater tendency to form polymers during protein folding than to form polymers from the folded protein. Our finding provides insights into biochemical approaches to treating serpinopathies by targeting a polymerogenic folding intermediate.     

Complete conformational stability of kinetically stable dimeric serine protease milin against pH,temperature, urea,and proteolysis

Spectroscopic, calorimetric, and proteolytic methods were utilized to evaluate the stability of the kinetically stable, differentially glycosylated, dimeric serine protease milin as a function of pH (1.0–11.0), temperature, urea, and GuHCl denaturation in presence of 8 M urea at pH 2.0. The stability of milin remains equivalent to that of native at pH 1.0–11.0. However, negligible and reversible alteration in structure upon temperature transition has been observed at pH 2.0 and with 1.6 M GuHCl. Irreversible and incomplete calorimetric transition with apparent T _m > 100°C was observed at basic pH (9.0 and 10.0). Urea-induced unfolding at pH 4.0, and at pH 2.0 with GuHCl, in presence of 8 M urea also reveals incomplete unfolding. Milin has been found to exhibit proteolytic resistant in either native or denatured state against various commercial proteases. These results imply that the high conformational stability of milin against various denaturating conditions enable its potential use in protease-based industries.     

The denatured state under native conditions: a non-native-like collapsed state of N-PGK

The guanidinium-denatured state of the N-domain of phosphoglycerate kinase (PGK) has been characterized using solution NMR. Rather than behaving as a homogenous ensemble of random coils, chemical shift changes for the majority of backbone amide resonances indicate that the denatured ensemble undergoes two definable equilibrium transitions upon titration with guanidinium, in addition to the major refolding event. (13)C and (15)N chemical shift changes indicate that both intermediary states have distinct helical character. At denaturant concentrations immediately above the mid-point of unfolding, size-exclusion chromatography shows N-PGK to have a compact, denatured form, suggesting that it forms a helical molten globule. Within this globule, the helices extend into some regions that become beta strands in the native state. This predisposition of the denatured state to extensive non-native-like conformation, illustrates that, rather than directing folding, conformational pre-organization in the denatured state can compete with the normal folding direction. The corresponding reduction in control of the direction of folding as proteins become larger, could thus constitute a restriction on the size of protein domains.     

Mutational Studies Uncover Non-Native Structure in the Dimeric Kinetic Intermediate of the H2A-H2B Heterodimer

The folding pathway of the histone H2A-H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I₂. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below ∼ 0.4 M urea (concentrations inaccessible from the  4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I₂ ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I₂ ensemble was assessed with a set of H2A-H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central α2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I₂ and the transition state between I₂ and N₂. This limited mutational study indicated that residues in the α2 helices of H2A and H2B as well as α1 of H2B and both the C-terminus of α3 and the short αC helix of H2A contribute to the stability of the I₂ burst-phase species. Interestingly, at least eight of the nine targeted residues stabilize I₂ by interactions that are non-native to some extent. Given that destabilizing I₂ and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structures present in the I₂ ensemble enable efficient folding of H2A-H2B.     

Structural analysis of kinetic folding intermediates for a TIM barrel protein, indole-3-glycerol phosphate synthase, by hydrogen exchange mass spectrometry and Gō model simulation

The structures of partially folded states appearing during the folding of a (βα)₈ TIM barrel protein, the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS), was assessed by hydrogen exchange mass spectrometry (HX-MS) and Gō model simulations. HX-MS analysis of the peptic peptides derived from the pulse-labeled product of the sub-millisecond folding reaction from the urea-denatured state revealed strong protection in the (βα)₄ region, modest protection in the neighboring (βα)_1-3 and (βα)₅β₆ segments and no significant protection in the remaining N and C-terminal segments. These results demonstrate that this species is not a collapsed form of the unfolded state under native-favoring conditions nor is it the native state formed via fast-track folding. However, the striking contrast of these results with the strong protection observed in the (βα)_2-5β₆ region after 5 s of folding demonstrates that these species represent kinetically distinct folding intermediates that are not identical as previously thought. A re-examination of the kinetic folding mechanism by chevron analysis of fluorescence data confirmed distinct roles for these two species: the burst-phase intermediate is predicted to be a misfolded, off-pathway intermediate, while the subsequent 5 s intermediate corresponds to an on-pathway equilibrium intermediate. Comparison with the predictions using a C^α Gō model simulation of the kinetic folding reaction for sIGPS shows good agreement with the core of the structure offering protection against exchange in the on-pathway intermediate(s). Because the native-centric Gō model simulations do not explicitly include sequence-specific information, the simulation results support the hypothesis that the topology of TIM barrel proteins is a primary determinant of the folding free energy surface for the productive folding reaction. The early misfolding reaction must involve aspects of non-native structure not detected by the Gō model simulation.     

Folding dynamics of phenylalanine hydroxylase depends on the enzyme’s metallation state: the native metal,iron, protects against aggregate intermediates

Phenylalanine hydroxylase (PAH), a non-heme iron enzyme, is responsible for the phenylalanine conversion to tyrosine. Its malfunction causes phenylketonuria (PKU). To better understand how protein structure and folding profiles are affected by the metal cofactor, we investigated the chemical (un)folding of apo- and holo-PAH from Chromobacterium violaceum (cPAH) using circular dichroism (CD) and analytical ultracentrifugation (AUC). Holo-cPAH shows a two-state unfolding transition. In contrast, the unfolding profile for apo-cPAH reveals a three-state (un)folding pathway and accumulation of an intermediate (apo-cPAH^I). This intermediate is also observed in refolding experiments. Fluorescence studies are consistent with the CD findings. The intermediate apo-cPAH^I and unfolded state(s) of apo- and holo-cPAH^U have been characterized by analytical ultracentrifugation (AUC). At 2.4 and 2.8 M GuHCl, 90% of the signal for apo-cPAH has a weight average sedimentation coefficient in water at 20°C (s20,w) of about 48 S, representing multiple aggregate species made of multiple monomers of cPAH. Aggregate formation for apo-cPAH is also confirmed by dynamic light scattering and electron microscopy giving a hydrodynamic radius (R_H) of 41 nm for apo-cPAH^I versus 3.5 nm for the native protein.     

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~ 27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency > 90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.     

Single-Molecule Studies of the Im7 Folding Landscape

Under appropriate conditions, the four-helical Im7 (immunity protein 7) folds from an ensemble of unfolded conformers to a highly compact native state via an on-pathway intermediate. Here, we investigate the unfolded, intermediate, and native states populated during folding using diffusion single-pair fluorescence resonance energy transfer by measuring the efficiency of energy transfer (or proximity or P ratio) between pairs of fluorophores introduced into the side chains of cysteine residues placed in the center of helices 1 and 4, 1 and 3, or 2 and 4. We show that while the native states of each variant give rise to a single narrow distribution with high P values, the distributions of the intermediates trapped at equilibrium (denoted I^eqm) are fitted by two Gaussian distributions. Modulation of the folding conditions from those that stabilize the intermediate to those that destabilize the intermediate enabled the distribution of lower P value to be assigned to the population of the unfolded ensemble in equilibrium with the intermediate state. The reduced stability of the I^eqm variants allowed analysis of the effect of denaturant concentration on the compaction and breadth of the unfolded state ensemble to be quantified from 0 to 6 M urea. Significant compaction is observed as the concentration of urea is decreased in both the presence and absence of sodium sulfate, as previously reported for a variety of proteins. In the presence of Na₂SO₄ in 0 M urea, the P value of the unfolded state ensemble approaches that of the native state. Concurrent with compaction, the ensemble displays increased peak width of P values, possibly reflecting a reduction in the rate of conformational exchange among iso-energetic unfolded, but compact conformations. The results provide new insights into the initial stages of folding of Im7 and suggest that the unfolded state is highly conformationally constrained at the outset of folding.     

Kinetic folding of Haloferax volcanii and Escherichia coli dihydrofolate reductases: haloadaptation by unfolded state destabilization at high ionic strength

Salts affect protein stability by multiple mechanisms (e.g., the Hofmeister effect, preferential hydration, electrostatic effects and weak ion binding). These mechanisms can affect the stability of both the native state and the unfolded state. Previous equilibrium stability studies demonstrated that KCl stabilizes dihydrofolate reductases (DHFRs) from Escherichia coli (ecDHFR, E. coli DHFR) and Haloferax volcanii (hvDHFR1, H. volcanii DHFR encoded by the hdrA gene) with similar efficacies, despite adaptation to disparate physiological ionic strengths (0.2 M versus 2 M). Kinetic studies can provide insights on whether equilibrium effects reflect native state stabilization or unfolded state destabilization. Similar kinetic mechanisms describe the folding of urea-denatured ecDHFR and hvDHFR1: a 5-ms stopped-flow burst-phase species that folds to the native state through two sequential intermediates with relaxation times of 0.1-3 s and 25-100 s. The latter kinetic step is very similar to that observed for the refolding of hvDHFR1 from low ionic strength. The unfolding of hvDHFR1 at low ionic strength is relatively slow, suggesting kinetic stabilization as observed for some thermophilic enzymes. Increased KCl concentrations slow the urea-induced unfolding of ecDHFR and hvDHFR1, but much less than expected from equilibrium studies. Unfolding rates extrapolated to 0 M denaturant, k_unf(H₂O), are relatively independent of ionic strength, demonstrating that the KCl-induced stabilization of ecDHFR and hvDHFR1 results predominantly from destabilization of the unfolded state. This supports the hypothesis from previous equilibrium studies that haloadaptation harnesses the effects of elevated salt concentrations on the properties of the aqueous solvent to enhance protein stability.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.     

Identification of a collapsed intermediate with non-native long-range interactions on the folding pathway of a pair of Fyn SH3 domain mutants by NMR relaxation dispersion spectroscopy

Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data.     

Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.     

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing (15)N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R(2) rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.     

The paradoxical behavior of a highly structured misfolded intermediate in RNA folding

Like many structured RNAs, the Tetrahymena group I ribozyme is prone to misfolding. Here we probe a long-lived misfolded species, referred to as M, and uncover paradoxical aspects of its structure and folding. Previous work indicated that a non-native local secondary structure, termed alt P3, led to formation of M during folding in vitro. Surprisingly, hydroxyl radical footprinting, fluorescence measurements with site-specifically incorporated 2-aminopurine, and functional assays indicate that the native P3, not alt P3, is present in the M state. The paradoxical behavior of alt P3 presumably arises because alt P3 biases folding toward M, but, after commitment to this folding pathway and before formation of M, alt P3 is replaced by P3. Further, structural and functional probes demonstrate that the misfolded ribozyme contains extensive native structure, with only local differences between the two states, and the misfolded structure even possesses partial catalytic activity. Despite the similarity of these structures, re-folding of M to the native state is very slow and is strongly accelerated by urea, Na+, and increased temperature and strongly impeded by Mg2+ and the presence of native peripheral contacts. The paradoxical observations of extensive native structure within the misfolded species but slow conversion of this species to the native state are readily reconciled by a model in which the misfolded state is a topological isomer of the native state, and computational results support the feasibility of this model. We speculate that the complex topology of RNA secondary structures and the inherent rigidity of RNA helices render kinetic traps due to topological isomers considerably more common for RNA than for proteins.