Folding and stability of the isolated Greek key domains of the long-lived human lens proteins gammaD-crystallin and gammaS-crystallin

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric gamma-crystallins and oligomeric beta-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human gammaD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human gammaD-crystallin, as well as the isolated domains of human gammaS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37 degrees C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human gammaD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein.     

High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-barrel fold

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K(D)) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel beta-barrel with the strand order (-beta1 -beta2 -beta3 -beta6 -beta5 -beta4 -beta7 -beta8), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.     

Interdomain interactions in hinge-bending transitions.

BACKGROUND: The mechanisms that allow or constrain protein movement have not been understood. Here we study interdomain interactions in proteins to investigate hinge-bending motions. RESULTS: We find a limited number of salt bridges and hydrogen bonds at the interdomain interface, in both the "closed" and the "open" conformations. Consistently, analysis of 222 salt bridges in an independently selected database indicates that most salt bridges form within rather than between independently folding hydrophobic units. Calculations show that these interdomain salt bridges either destabilize or only marginally stabilize the closed conformation in most proteins. In contrast, the nonpolar buried surface area between the moving parts can be extensive in the closed conformations. However, when the nonpolar buried surface area is large, we find that at the interdomain interface in the open conformation it may be as large or larger than in the closed conformation. Hence, the energetic penalty of opening the closed conformation is overcome. Consistently, a large nonpolar surface area buried in the closed interdomain interface accompanies limited opening of the domains, yielding a larger interface. CONCLUSIONS: Short-range electrostatic interactions are largely absent between moving domains. Interdomain nonpolar buried surface area may be large in the closed conformation, but it is largely offset by the area buried in the open conformation. In such cases the opening of the domains appears to be relatively small. This may allow prediction of the extent of domain opening. Such predictions may have implications for the shape and size of the binding pockets in drug/protein design.     

Stability and unfolding of reduced Escherichia coli glutaredoxin 2: a monomeric structural homologue of the glutathione transferase family

Glutaredoxin 2 (Grx2) from Escherichia coli is monomeric and an atypical glutaredoxin that takes part in the monothiol deglutathionylation of proteins. Unlike its orthologs, Grx2 is a larger molecule with a canonical glutathione transferase (GST) fold that consists of two structurally distinct domains, an N-terminal glutaredoxin domain and a C-terminal alpha-helical domain. While GSTs are dimeric proteins, the conformational stability and unfolding kinetics of Grx2 were investigated to establish the contribution made by the domain interface to the stability of the tertiary structure of GST-like proteins without any influence from quaternary interactions. Equilibrium unfolding transitions for Grx2, using urea as a denaturant, are monophasic and exhibit coincidence of the fluorescence and CD data indicative of a concerted loss or formation of tertiary and secondary structure. The data fit well to a two-state N <--> U model with no evidence that an intermediate is being formed. The experimental m value [2.7 kcal mol (-1) (M urea) (-1)] is in excellent agreement with a predicted value of 2.5 kcal mol (-1) (M urea) (-1) based on the amount of surface area expected to become exposed during unfolding. These findings provide evidence that the two structurally distinct domains of Grx2 behave as a single cooperative folding unit. The unfolding kinetics are complex which, as a result of native-state heterogeneity, are characterized by two observable unfolding reactions that occur in parallel. A major population representing one distinct nativelike form unfolds on a fast track to denatured Grx2 with cis-Pro49. This is followed by a spectroscopically silent cis-trans proline isomerization reaction as determined by interrupted unfolding experiments. A minor population representing the other distinct nativelike form unfolds slowly with its rate being limited by an undetermined structural isomerization reaction. Further, there is no evidence indicating that unfolding proceeds via a high-energy intermediate that might suggest independent unfolding of the two nonidentical domains in Grx2. The kinetics data are, therefore, consistent with the existence of cooperativity between the domains, in agreement with the equilibrium data.     

Contributions of hydrophobic domain interface interactions to the folding and stability of human gammaD-crystallin

Human gammaD-crystallin (HgammaD-Crys) is a monomeric eye lens protein composed of two highly homologous beta-sheet domains. The domains interact through interdomain side chain contacts forming two structurally distinct regions, a central hydrophobic cluster and peripheral residues. The hydrophobic cluster contains Met43, Phe56, and Ile81 from the N-terminal domain (N-td) and Val132, Leu145, and Val170 from the C-terminal domain (C-td). Equilibrium unfolding/refolding of wild-type HgammaD-Crys in guanidine hydrochloride (GuHCl) was best fit to a three-state model with transition midpoints of 2.2 and 2.8 M GuHCl. The two transitions likely corresponded to sequential unfolding/refolding of the N-td and the C-td. Previous kinetic experiments revealed that the C-td refolds more rapidly than the N-td. We constructed alanine substitutions of the hydrophobic interface residues to analyze their roles in folding and stability. After purification from E. coli, all mutant proteins adopted a native-like structure similar to wild type. The mutants F56A, I81A, V132A, and L145A had a destabilized N-td, causing greater population of the single folded domain intermediate. Compared to wild type, these mutants also had reduced rates for productive refolding of the N-td but not the C-td. These data suggest a refolding pathway where the domain interface residues of the refolded C-td act as a nucleating center for refolding of the N-td. Specificity of domain interface interactions is likely important for preventing incorrect associations in the high protein concentrations of the lens nucleus.     

A conserved "hydrophobic staple motif" plays a crucial role in the refolding of human glutathione transferase P1-1

The specific (i, i+5) hydrophobic staple interaction involving a helix residue and a second residue located in the turn preceding the helix is a recurrent motif at the N terminus of alpha-helices. This motif is strictly conserved in the core of all soluble glutathione transferases (GSTs) as well as in other protein structures. Human GSTP1-1 variants mutated in amino acid Ile(149) and Tyr(154) of the hydrophobic staple motif of the alpha6-helix were analyzed. In particular, a double mutant cycle analysis has been performed to evaluate the role of the hydrophobic staple motif in the refolding process. The results show that this local interaction, by restricting the number of conformations of the alpha6-helix relative to the alpha1-helix, favors the formation of essential interdomain interactions and thereby accelerates the folding process. Thus, for the first time it is shown that the hydrophobic staple interaction has a role in the folding process of an intact protein. In P(i) class GSTs, Tyr(154) appears to be of particular structural importance, since it interacts with conserved residues Leu(21), Asp(24), and Gln(25) of the adjacent alpha1-helix which contributes to the active site. Human GSTP1-1 variants L21A and Y154F have also been analyzed in order to distinguish the role of interdomain interactions from that of the hydrophobic staple. The experimental results reported here suggest that the strict conservation of the hydrophobic staple motif reflects an evolutionary pressure for proteins to fold rapidly.     

The folding and evolution of multidomain proteins

Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent. Furthermore, recent studies indicate that multidomain proteins have evolved mechanisms to minimize the problems of interdomain misfolding.     

Cooperative folding in a multi-domain protein

Most protein domains are found in multi-domain proteins, yet most studies of protein folding have concentrated on small, single-domain proteins or on isolated domains from larger proteins. Spectrin domains are small (106 amino acid residues), independently folding domains consisting of three long alpha-helices. They are found in multi-domain proteins with a number of spectrin domains in tandem array. Structural studies have shown that in these arrays the last helix of one domain forms a continuous helix with the first helix of the following domain. It has been demonstrated that a number of spectrin domains are stabilised by their neighbours. Here we investigate the molecular basis for cooperativity between adjacent spectrin domains 16 and 17 from chicken brain alpha-spectrin (R16 and R17). We show that whereas the proteins unfold as a single cooperative unit at 25 degrees C, cooperativity is lost at higher temperatures and in the presence of stabilising salts. Mutations in the linker region also cause the cooperativity to be lost. However, the cooperativity does not rely on specific interactions in the linker region alone. Most mutations in the R17 domain cause a decrease in cooperativity, whereas proteins with mutations in the R16 domain still fold cooperatively. We propose a mechanism for this behaviour.     

Probing possible downhill folding: native contact topology likely places a significant constraint on the folding cooperativity of proteins with approximately 40 residues

Experiments point to appreciable variations in folding cooperativity among natural proteins with approximately 40 residues, indicating that the behaviors of these proteins are valuable for delineating the contributing factors to cooperative folding. To explore the role of native topology in a protein's propensity to fold cooperatively and how native topology might constrain the degree of cooperativity achievable by a given set of physical interactions, we compared folding/unfolding kinetics simulated using three classes of native-centric C^α chain models with different interaction schemes. The approach was applied to two homologous 45-residue fragments from the peripheral subunit-binding domain family and a 39-residue fragment of the N-terminal domain of ribosomal protein L9. Free-energy profiles as functions of native contact number were computed to assess the heights of thermodynamic barriers to folding. In addition, chevron plots of folding/unfolding rates were constructed as functions of native stability to facilitate comparison with available experimental data. Although common Gō-like models with pairwise Lennard-Jones-type interactions generally fold less cooperatively than real proteins, the rank ordering of cooperativity predicted by these models is consistent with experiment for the proteins investigated, showing increasing folding cooperativity with increasing nonlocality of a protein's native contacts. Models that account for water-expulsion (desolvation) barriers and models with many-body (nonadditive) interactions generally entail higher degrees of folding cooperativity indicated by more linear model chevron plots, but the rank ordering of cooperativity remains unchanged. A robust, experimentally valid rank ordering of model folding cooperativity independent of the multiple native-centric interaction schemes tested here argues that native topology places significant constraints on how cooperatively a protein can fold.     

The function of interdomain interactions in controlling nucleotide exchange rates in transducin

The intramolecular contacts in heterotrimeric G proteins that determine the rates of basal and receptor-stimulated nucleotide exchange are not fully understood. The alpha subunit of heterotrimeric G proteins consists of two domains: a Ras-like domain with structural homology to the monomeric G protein Ras and a helical domain comprised of six alpha-helices. The bound nucleotide lies in a deep cleft between the two domains. Exchange of the bound nucleotide may involve opening of this cleft. Thus interactions between the domains may affect the rate of nucleotide exchange in G proteins. We have tested this hypothesis in the alpha subunit of the rod cell G protein transducin (Galpha(t)). Site-directed mutations were prepared in a series of residues located at the interdomain interface. The proteins were expressed in vitro in a reticulocyte lysate system. The rates of basal and rhodopsin-catalyzed nucleotide exchange were determined using a trypsin digestion assay specifically adapted for kinetic measurements. Charge-altering substitutions of two residues at the interdomain interface, Lys(273) and Lys(276), increased basal nucleotide exchange rates modestly (5-10-fold). However, we found no evidence that interactions spanning the two domains in Galpha(t) significantly affected either basal or rhodopsin-catalyzed nucleotide exchange rates. These results suggest that opening of the interdomain cleft is not an energetic barrier to nucleotide exchange in Galpha(t). Experiments with Galpha(i1) suggest by comparison that the organization and function of the interdomain region differ among various G protein subtypes.     

Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force.

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.     

Mutational analysis of hydrophobic domain interactions in gamma B-crystallin from bovine eye lens.

gamma B-crystallin is a monomeric member of the beta gamma-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all-beta Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one- or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner. It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete gamma B-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for gamma B wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of gamma B-crystallin.     

Kinetics of folding nuclei formation in proteins

When a protein folds or unfolds, it passes through many half-folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed in the vicinity of the equilibrium point. Here the "two-state" transition proceeds without any accumulation of metastable intermediates, and only the transition state ("folding nucleus") is outlined by its key influence on the folding/unfolding kinetics. This review covers recent experimental and theoretical studies of folding nuclei.     

In the Multi-domain Protein Adenylate Kinase,Domain Insertion Facilitates Cooperative Folding while Accommodating Function at Domain Interfaces

Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein, E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.     

X-ray structures of three interface mutants of gammaB-crystallin from bovine eye lens.

GammaB-crystallin consists of two domains each comprising two "Greek key" motifs. Both domains fold independently, and domain interactions contribute significantly to the stability of the C-terminal domain. In a previous study (Palme S et al., 1996, Protein Sci 6:1529-1636) it was shown that Phe56 from the N-terminal domain, a residue involved in forming a hydrophobic core at the domain interface, effects the interaction of the two domains, and therefore, the stability of the C-terminal domain. Ala or Asp at position 56 drastically decreased the stability of the C-terminal domain, whereas Trp had a more moderate effect. In this article we present the X-ray structures of these interface mutants and correlate them with the stability data. The mutations do not effect the overall structure of the molecule. No structural changes are observed in the vicinity of the replaced residue, suggesting that the local structure is too rigid to allow compensations for the amino acid replacements. In the mutants gammaB-F56A and -F56D, a solvent-filled groove accessible to the bulk solvent is created by the replacement of the bulky Phe side chain. In gammaB-F56W, the pyrrole moiety of the indole ring replaces the phenyl side chain of the wild type. With the exception of gammaB-F56W, there is a good correlation between the hydrophobicity of the amino acid at position 56 according to the octanol scale and the stability of the C-terminal domain. In gammaB-F56W, the C-terminal domain is less stable than estimated from the hydrophobicity, presumably because the ring nitrogen (Nepsilon1) has no partner to form hydrogen bonds. The data suggest that the packing of hydrophobic residues in the interface core is important for domain interactions and the stability of gammaB-crystallin. Apparently, for protein stability, the same principles apply for hydrophobic cores within domains and at domain interfaces.     

Structural basis of the unusual stability and substrate specificity of ervatamin C, a plant cysteine protease from Ervatamia coronaria

Ervatamin C is an unusually stable cysteine protease from the medicinal plant Ervatamia coronaria belonging to the papain family. Though it cleaves denatured natural proteins with high specific activity, its activity toward some small synthetic substrates is found to be insignificant. The three-dimensional structure and amino acid sequence of the protein have been determined from X-ray diffraction data at 1.9 A (R = 17.7% and R(free) = 19.0%). The overall structure of ervatamin C is similar to those of other homologous cysteine proteases of the family, folding into two distinct left and right domains separated by an active site cleft. However, substitution of a few amino acid residues, which are conserved in the other members of the family, has been observed in both the domains and also at the region of the interdomain cleft. Consequently, the number of intra- and interdomain hydrogen-bonding interactions is enhanced in the structure of ervatamin C. Moreover, a unique disulfide bond has been identified in the right domain of the structure, in addition to the three conserved disulfide bridges present in the papain family. All these factors contribute to an increase in the stability of ervatamin C. In this enzyme, the nature of the S2 subsite, which is the primary determinant of specificity of these proteases, is similar to that of papain, but at the S3 subsite, Ala67 replaces an aromatic residue, and has the effect of eliminating sufficient hydrophobic interactions required for S3-P3 stabilization. This provides the possible explanation for the lower activity of ervatamin C toward the small substrate/inhibitor. This substitution, however, does not affect the binding of denatured natural protein substrates to the enzyme significantly, as there exist a number of additional interactions at the enzyme-substrate interface outside the active site cleft.     

Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate

Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central alpha-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with DeltaG degrees (H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.     

Pathway modulation, circular permutation and rapid RNA folding under kinetic control

The thermodynamics and folding kinetics of a circularly permuted construct of the ribozyme from Bacillus subtilis RNase P are analyzed and compared with the folding properties of the wild-type ribozyme using optical spectroscopy and catalytic activity. The folding of the wild-type ribozyme is slow due to the rearrangement of kinetically trapped species containing misfolded structures. To test whether any misfolded structure arises from interactions between the two independently folding domains of the RNase P RNA, a circular permuted form was created where one of the two phosphodiester bonds connecting these domains is broken. This construct folds approximately 15-fold faster (t1/2 approximately nine seconds) than the wild-type ribozyme at 37 degreesC. While the complete folding of both domains is kinetically indistinguishable in the wild-type ribozyme, one domain folds much faster than the other domain in the circularly permuted construct. Hence, the major kinetic trap in the folding of the wild-type RNase P RNA involves interdomain interactions. This kinetic trap is avoidable at 37 degreesC in the circularly permuted RNA. However, at temperatures below 30 degreesC or when refolding begins from an equilibrium intermediate stabilized by submillimolar concentrations of Mg2+, a subpopulation containing an interdomain misfold still forms. These results indicate that the folding pathway of this large RNA is highly malleable and can be under kinetic control.