Protein Folding: Adding a Nucleus to Guide Helix Docking Reduces Landscape Roughness

The elongated three-helix‐bundle spectrin domains R16 and R17 fold and unfold unusually slowly over a rough energy landscape, in contrast to the homologue R15, which folds fast over a much smoother, more typical landscape. R15 folds via a nucleation–condensation mechanism that guides the docking of the A and C-helices. However, in R16 and R17, the secondary structure forms first and the two helices must then dock in the correct register. Here, we use variants of R16 and R17 to demonstrate that substitution of just five key residues is sufficient to alter the folding mechanism and reduce the landscape roughness. We suggest that, by providing access to an alternative, faster, folding route over their landscape, R16 and R17 can circumvent their slow, frustrated wild-type folding mechanism.     

Different Members of a Simple Three-Helix Bundle Protein Family Have Very Different Folding Rate Constants and Fold by Different Mechanisms

The 15th, 16th, and 17th repeats of chicken brain α-spectrin (R15, R16, and R17, respectively) are very similar in terms of structure and stability. However, R15 folds and unfolds 3 orders of magnitude faster than R16 and R17. This is unexpected. The rate-limiting transition state for R15 folding is investigated using protein engineering methods (Φ-value analysis) and compared with previously completed analyses of R16 and R17. Characterisation of many mutants suggests that all three proteins have similar complexity in the folding landscape. The early rate-limiting transition states of the three domains are similar in terms of overall structure, but there are significant differences in the patterns of Φ-values. R15 apparently folds via a nucleation-condensation mechanism, which involves concomitant folding and packing of the A- and C-helices, establishing the correct topology. R16 and R17 fold via a more framework-like mechanism, which may impede the search to find the correct packing of the helices, providing a possible explanation for the fast folding of R15.     

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.     

The folding pathway of a single domain in a multidomain protein is not affected by its neighbouring domain

Domains are the structural, functional, and evolutionary components of proteins. Most folding studies to date have concentrated on the folding of single domains, but more than 70% of human proteins contain more than one domain, and interdomain interactions can affect both the stability and the folding kinetics. Whether the folding pathway is altered by interdomain interactions is not yet known. Here we investigated the effect of a folded neighbouring domain on the folding pathway of spectrin R16 (the 16th α-helical repeat from chicken brain α-spectrin) by using the two-domain construct R1516. The R16 folds faster and unfolds more slowly in the presence of its folded neighbour R15 (the 15th α-helical repeat from chicken brain α-spectrin). An extensive Φ-value analysis of the R16 domain in R1516 was completed to compare the transition state of the R16 domain alone with that of the R16 domain in a multidomain construct. The results indicate that the folding pathways are the same. This result validates the current approach of breaking up larger proteins into domains for the study of protein folding pathways.     

Probing the Effects of Local Frustration in the Folding of a Multidomain Protein

Our current knowledge of protein folding is primarily based on experimental data obtained from isolated domains. In fact, because of their complexity, multidomain proteins have been elusive to the experimental characterization. Thus, the folding of a domain in isolation is generally assumed to resemble what should be observed for more complex structural architectures. Here we compared the folding mechanism of a protein domain in isolation and in the context of its supramodular multidomain structure. By carrying out an extensive mutational analysis we illustrate that while the early events of folding are malleable and influenced by the absence/presence of the neighboring structures, the late events appear to be more robust. These effects may be explained by analyzing the local frustration patterns of the domain, providing critical support for the funneled energy landscape theory of protein folding, and highlighting the role of protein frustration in sculpting the early events of the reaction.     

Protein folding forces

We investigate the average inter-residue folding forces derived from mutational data of the 15 proteins: barstar, barnase, chymotrypsin inhibitor 2 (CI2), Src SH3 domain, spectrin R16 domain, Arc repressor, apo-azurin, cold shock protein B (cspB), C-terminal domain of ribosomal protein L9 (CTL9), FKBP12, α-lactalbumin, colicin E7 immunity protein 7 (IM7), colicin E9 immunity protein 9 (IM9), spectrin R17 domain, and ubiquitin. The residue-specific contributions to folding in most of the 15 protein molecules are highly non-uniformly distributed and are typically about 1 piconewton (pN) per interaction. The strongest folding forces often occur in some of the helices and strands of folding nuclei which suggests that folding nucleation−condensation is partially directed by formation of some secondary structure interactions. The correlation of the energy changes of mutants with inter-residue contact maps of the protein molecules provides a higher resolution than assigning the mutant data to certain positions in the polypeptide strand alone. In contrast to previous Φ-value analysis, we now can partially resolve folding motions. Compaction of at least one α-helix along its axis mediated by internal hydrogen bonds and stabilized by diffuse tertiary structure interactions appears to be one important molecular event during early folding in barstar, CI2, spectrin R16 domain, Arc repressor, α-lactalbumin, IM7, IM9, and spectrin R17 domain. A lateral movement of at least two strands neighbored in sequence towards each other appears to be involved in early folding of the SH3 domain, cspB, CTL9, and FKBP12.     

Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate.

The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans----cis reisomerization of the Tyr-38-Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans----cis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate rate-limiting steps by trapping partially folded structures.     

Complex folding kinetics of a multidomain protein

Spectrin domains are three-helix bundles, commonly found in large tandem arrays. Equilibrium studies have shown that spectrin domains are significantly stabilized by their neighbors. In this work we show that domain:domain interactions can also have profound effects on their kinetic behavior. We have studied the folding of a tandem pair of spectrin domains (R1617) using a combination of single- and double-jump stopped flow experiments (monitoring folding by both circular dichroism and fluorescence). Mutant proteins were also used to investigate the complex folding kinetics. We find that, although the domains fold and unfold individually, there is a single rate-determining step for both folding and unfolding of the protein. This is consistent with the equilibrium observation of cooperative folding of the entire two-domain protein. The results may have important biological implications. Not only will the protein fold more efficiently during cotranslational folding, but the ability of the multidomain protein to withstand thermal unfolding in the cell will be dramatically increased. This study suggests that caution has to be exercised when extrapolating from single domains to larger proteins with a number of independently folding modules arranged in tandem. The multidomain protein spectrin is certainly more than "the sum of its parts".     

The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family

Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Φ-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Φ-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Φ values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Φ values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains.     

Distinguishing specific and nonspecific interdomain interactions in multidomain proteins

Multidomain proteins account for over two-thirds of the eukaryotic genome. Although there have been extensive studies into the biophysical properties of isolated domains, few have investigated how the domains interact. Spectrin is a well-characterized multidomain protein with domains linked in tandem array by contiguous helices. Several of these domains have been shown to be stabilized by their neighbors. Until now, this stabilization has been attributed to specific interactions between the natural neighbors, however we have recently observed that nonnatural neighboring domains can also induce a significant amount of stabilization. Here we investigate this nonnative stabilizing effect. We created spectrin-titin domain pairs of both spectrin R16 and R17 with a single titin I27 domain at either the N- or the C-terminus and found that spectrin domains are significantly stabilized, through slowed unfolding, by nonnative interactions at the C-terminus only. Of particular importance, we show that specific interactions between natural folded neighbors at either terminus confer even greater stability by additionally increasing the folding rate constants. We demonstrate that it is possible to distinguish between natural stabilizing interactions and nonspecific stabilizing effects through examination of the kinetics of well chosen mutant proteins. This work adds to the complexity of studying multidomain proteins.     

Spectrin domains lose cooperativity in forced unfolding

Spectrin is a multidomain cytoskeletal protein, the component three-helix bundle domains are expected to experience mechanical force in vivo. In thermodynamic and kinetic studies, neighboring domains of chicken brain alpha-spectrin R16 and R17 have been shown to behave cooperatively. Is this cooperativity maintained under force? The effect of force on these spectrin domains was investigated using atomic force microscopy. The response of the individual domains to force was compared to that of the tandem repeat R1617. Importantly, nonhelical linkers (all-beta immunoglobulin domains) were used to avoid formation of nonnative helical linkers. We show that, in contrast to previous studies on spectrin repeats, only 3% of R1617 unfolding events gave an increase in contour length consistent with cooperative two-domain unfolding events. Furthermore, the unfolding forces for R1617 were the same as those for the unfolding of R16 or R17 alone. This is a strong indication that the cooperative unfolding behavior observed in the stopped-flow studies is absent between these spectrin domains when force is acting as a denaturant. Our evidence suggests that the rare double unfolding events result from misfolding between adjacent repeats. We suggest that this switch from cooperative to independent behavior allows multidomain proteins to maintain integrity under applied force.     

Folding of ribonuclease T1. 1. Existence of multiple unfolded states created by proline isomerization

It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.     

Thermal stability of chicken brain α-spectrin repeat 17: a spectroscopic study

Spectrin is a rod-like multi-modular protein that is mainly composed of triple-helical repeats. These repeats show very similar 3D-structures but variable conformational and thermodynamical stabilities, which may be of great importance for the flexibility and dynamic behaviour of spectrin in the cell. For instance, repeat 17 (R17) of the chicken brain spectrin α-chain is four times less stable than neighbouring repeat 16 (R16) in terms of ?G. The structure of spectrin repeats has mainly been investigated by X-ray crystallography, but the structures of a few repeats, e.g. R16, have also been determined by NMR spectroscopy. Here, we undertook a detailed characterization of the neighbouring R17 by NMR spectroscopy. We assigned most backbone resonances and observed NOE restraints, relaxation values and coupling constants that all indicated that the fold of R17 is highly similar to that of R16, in agreement with previous X-ray analysis of a tandem repeat of the two domains. However, (15)N heteronuclear NMR spectra measured at different temperatures revealed particular features of the R17 domain that might contribute to its lower stability. Conformational exchange appeared to alter the linker connecting R17 to R16 as well as the BC-loop in close proximity. In addition, heat-induced splitting was observed for backbone resonances of a few spatially related residues including V99 of helix C, which in R16 is replaced by the larger hydrophobic tryptophan residue that is relatively conserved among other spectrin repeats. These data support the view that the substitution of tryptophan by valine at this position may contribute to the lower stability of R17.     

Folding of pig gastric mucin non-glycosylated domains: a discrete molecular dynamics study

Mucin glycoproteins consist of tandem-repeating glycosylated regions flanked by non-repetitive protein domains with little glycosylation. These non-repetitive domains are involved in polymerization of mucin and play an important role in the pH-dependent gelation of gastric mucin, which is essential for protecting the stomach from autodigestion. We examine folding of the non-repetitive sequence of PGM-2X (242 amino acids) and the von Willebrand factor vWF-C1 domain (67 amino acids) at neutral and low pH using discrete molecular dynamics (DMD) in an implicit solvent combined with a four-bead peptide model. Using the same implicit solvent parameters, folding of both domains is simulated at neutral and low pH. In contrast to vWF-C1, PGM-2X folding is strongly affected by pH as indicated by changes in the contact order, radius of gyration, free-energy landscape, and the secondary structure. Whereas the free-energy landscape of vWF-C1 shows a single minimum at both neutral and low pH, the free-energy landscape of PGM-2X is characterized by multiple minima that are more numerous and shallower at low pH. Detailed structural analysis shows that PGM-2X partially unfolds at low pH. This partial unfolding is facilitated by the C-terminal region GLU236-PRO242, which loses contact with the rest of the domain due to effective “mean-field” repulsion among highly positively charged N- and C-terminal regions. Consequently, at low pH, hydrophobic amino acids are more exposed to the solvent. In vWF-C1, low pH induces some structural changes, including an increased exposure of CYS at position 67, but these changes are small compared to those found in PGM-2X. For PGM-2X, the DMD-derived average β-strand propensity increases from 0.26 ± 0.01 at neutral pH to 0.38 ± 0.01 at low pH. For vWF-C1, the DMD-derived average β-strand propensity is 0.32 ± 0.02 at neutral pH and 0.35 ± 0.02 at low pH. The DMD-derived structural information provides insight into pH-induced changes in the folding of two distinct mucin domains and suggests plausible mechanisms of the aggregation/gelation of mucin.     

Signal Peptide-rheostat Dynamics Delay Secretory Preprotein Folding

Sec secretory proteins are distinguished from cytoplasmic ones by N-terminal signal peptides with multiple roles during post-translational translocation. They contribute to preprotein targeting to the translocase by slowing down folding, binding receptors and triggering secretion. While signal peptides get cleaved after translocation, mature domains traffic further and/or fold into functional states. How signal peptides delay folding temporarily, to keep mature domains translocation-competent, remains unclear. We previously reported that the foldon landscape of the periplasmic prolyl-peptidyl isomerase is altered by its signal peptide and mature domain features. Here, we reveal that the dynamics of signal peptides and mature domains crosstalk. This involves the signal peptide’s hydrophobic helical core, the short unstructured connector to the mature domain and the flexible rheostat at the mature domain N-terminus. Through this cis mechanism the signal peptide delays the formation of early initial foldons thus altering their hierarchy and delaying mature domain folding. We propose that sequence elements outside a protein’s native core exploit their structural dynamics to influence the folding landscape.     

Folding of tandem-linked domains

One of the factors, which influences protein folding in vivo, is a linkage of protein domains into multidomain tandems. However, relatively little is known about the impact of domain connectivity on protein folding mechanisms. In this article, we use coarse grained models of proteins to explore folding of tandem-linked domains (TLD). We found TLD folding to follow two scenarios. In the first, the tandem connectivity produces relatively minor impact on folding and the mechanisms of folding of tandem-linked and single domains remain similar. The second scenario involves qualitative changes in folding mechanism because of tandem linkage. As a result, protein domains, which fold via two-state mechanism as single isolated domains, may form new stable intermediates when inserted into tandems. The new intermediates are created by topological constraints imposed by the linkers between domains. In both cases tandem linkage slows down folding. We propose that the impact of tandem connectivity can be minimized, if the terminal secondary structure elements (SSEs) are flexible. In particular, two factors appear to facilitate TLD folding: (1) the interactions between terminal SSE are poorly ordered in the folding transition state, whereas nonterminal SSE are better structured, (2) the interactions between terminal SSE are weak in the native state. We apply these findings to wild-type proteins by examining experimental phi-value data and by performing all-atom molecular dynamics simulations. We show that immunoglobulin-like domains appear to utilize the factors, which minimize the impact of tandem connectivity on their folding. Several single domain proteins, which are likely to misfold in tandems, are also identified.     

Folding and Misfolding of a PDZ Tandem Repeat

Although the vast majority of the human proteome is represented by multi-domain proteins, the study of multi-domain folding and misfolding is a relatively poorly explored field. The protein Whirlin is a multi-domain scaffolding protein expressed in the inner ear. It is characterized by the presence of tandem repeats of PDZ domains. The first two PDZ domains of Whirlin (PDZ1 and PDZ2 – namely P1P2) are structurally close and separated by a disordered short linker. We recently described the folding mechanism of the P1P2 tandem. The difference in thermodynamic stability of the two domains allowed us to selectively unfold one or both PDZ domains and to pinpoint the accumulation of a misfolded intermediate, which we demonstrated to retain physiological binding activity. In this work, we provide an extensive characterization of the folding and unfolding of P1P2. Based on the observed data, we describe an integrated kinetic analysis that satisfactorily fits the experiments and provides a valuable model to interpret multi-domain folding. The experimental and analytical approaches described in this study may be of general interest for the interpretation of complex multi-domain protein folding kinetics.     

Active Cage Mechanism of Chaperonin-Assisted Protein Folding Demonstrated at Single-Molecule Level

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli have an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations (100 pM) to exclude aggregation, we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP) by single-pair fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We find that GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. Accelerated folding is achieved by encapsulation of folding intermediate in the GroEL/ES cage, independent of repetitive cycles of protein binding and release from GroEL. Moreover, photoinduced electron transfer experiments provided direct physical evidence that the confining environment of the chaperonin restricts polypeptide chain dynamics. This effect is mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. EL(KKK2)/ES functions as a passive cage in which folding occurs at the slow spontaneous rate. Taken together our findings suggest that protein encapsulation can accelerate folding by entropically destabilizing folding intermediates, in strong support of an active chaperonin mechanism in the folding of some proteins. Accelerated folding is biologically significant as it adjusts folding rates relative to the speed of protein synthesis.     

Mechanism of Assembly of the Non-Covalent Spectrin Tetramerization Domain from Intrinsically Disordered Partners

Interdomain interactions of spectrin are critical for maintenance of the erythrocyte cytoskeleton. In particular, “head-to-head” dimerization occurs when the intrinsically disordered C-terminal tail of β-spectrin binds the N-terminal tail of α-spectrin, folding to form the “spectrin tetramer domain”. This non-covalent three-helix bundle domain is homologous in structure and sequence to previously studied spectrin domains. We find that this tetramer domain is surprisingly kinetically stable. Using a protein engineering Φ-value analysis to probe the mechanism of formation of this tetramer domain, we infer that the domain folds by the docking of the intrinsically disordered β-spectrin tail onto the more structured α-spectrin tail.     

The Plastic Energy Landscape of Protein Folding: A TRIANGULAR FOLDING MECHANISM WITH AN EQUILIBRIUM INTERMEDIATE FOR A SMALL PROTEIN DOMAIN*

Protein domains usually fold without or with only transiently populated intermediates, possibly to avoid misfolding, which could result in amyloidogenic disease. Whether observed intermediates are productive and obligatory species on the folding reaction pathway or dispensable by-products is a matter of debate. Here, we solved the crystal structure of a small protein domain, SAP97 PDZ2 I342W C378A, and determined its folding pathway. The presence of a folding intermediate was demonstrated both by single and double-mixing kinetic experiments using urea-induced (un)folding as well as ligand-induced folding. This protein domain was found to fold via a triangular scheme, where the folding intermediate could be either on- or off-pathway, depending on the experimental conditions. Furthermore, we found that the intermediate was present at equilibrium, which is rarely seen in folding reactions of small protein domains. The folding mechanism observed here illustrates the roughness and plasticity of the protein folding energy landscape, where several routes may be employed to reach the native state. The results also reconcile the folding mechanisms of topological variants within the PDZ domain family.