Structural Determinants of Misfolding in Multidomain Proteins
Protein misfolding and aggregation are well-known for their association with amyloidosis and other diseases 1 , 2 ., Proteins with two or more domains are abundant in higher organisms , accounting for up to 70% of all eukaryotic proteins , and domain-repeat proteins in particular occupy a fraction up to 20% of the proteomes in multicellular organisms 3 , 4 , therefore their folding is of considerable relevance 5 ., Since there is often some sequence similarity between domains with the same structure , it is easily possible to imagine that multidomain proteins containing repeats of domains with the same fold might be susceptible to misfolding ., Indeed , misfolding of multidomain proteins has been observed in many protein families 6 ., Single molecule techniques have been particularly powerful for studying folding/misfolding of such proteins , in particular Förster resonance energy transfer ( FRET ) and atomic force microscopy ( AFM ) ., For instance , recent studies using single-molecule FRET , in conjunction with coarse-grained simulations , have revealed the presence of domain-swapped misfolded states in tandem repeats of the immunoglobulin-like domain I27 from the muscle protein Titin 7 ( an example is shown in Fig 1e ) ., Domain-swapping 2 involves the exchange of secondary structure elements between two protein domains with the same structure ., Remarkably , these misfolded states are stable for days , much longer than the unfolding time of a single Titin domain ., The domain-swapped misfolds identified in the Titin I27 domains are also consistent with earlier observations of misfolding in the same protein by AFM , although not given a structural interpretation at the time 8 ., In addition , AFM experiments have revealed what appears to be a similar type of misfolding in polyproteins consisting of eight tandem repeats of the same fibronectin type III domain from tenascin ( TNfn3 ) 9 , as well as in native constructs of tenascin 8 , and between the N-terminal domains of human γD-crystallin when linked in a synthetic oligomer 10 ., In addition to domain-swapped misfolding , an alternative type of misfolded state is conceivable for polyproteins in which the sequences of adjacent domains are similar , namely the formation of amyloid-like species with parallel β-sheets ., Theoretical work in fact made the prediction that such species would be formed in tandem repeats of titin domains 11 ., Recently , time-resolved single-molecule FRET experiments on tandem domains of I27 have revealed a surprising number of intermediates formed at short times , which include an unexpected species that appears to be consistent with the previously suggested amyloid-like state 12 ., However , since only the domain-swapped species persisted till long times , and therefore are the most likely to be problematic in cells , we focus on their formation in this work ., A simplified illustration of the mechanism for folding and misfolding , based on both coarse-grained simulations as well as single-molecule and ensemble kinetics 7 , 12 , is shown in Fig 1 , using the Titin I27 domain as an example ., Starting from the completely unfolded state in Fig 1a , correct folding would proceed via an intermediate in which either one of the domains is folded ( Fig 1b ) , and finally to the fully folded state , Fig 1c ., The domain-swapped misfolded state , an example of which is shown in Fig 1e , consists of two native-like folds which are in fact assembled by swapping of sequence elements from the N- and C-terminal portions of the protein ., The final structure in Fig 1e comprises what we shall refer to as a “central domain” formed by the central regions of the sequence ( on the left in Fig 1e ) and a “terminal domain” formed from the N- and C-termini ( on the right ) ., The intermediate structure in Fig 1d , suggested by coarse-grained simulations 7 , and supported by experiment 12 , has only the central domain folded ., This central domain can itself be viewed as a circular permutant 13 of the original native Titin I27 structure , as discussed further below ., While domain-swapped misfolding of tandem repeats has been identified in a number of proteins to date , there are several other proteins for which it does not occur to a detectable level ., For instance , extensive sampling of repeated unfolding and folding of a polyprotein of Protein G ( GB1 ) by AFM revealed no indication of misfolded states , in contrast to Titin 14 ., Similarly , early AFM studies on polyUbiquitin also did not suggest misfolded intermediates in constant force unfolding 15–20 , and lock-in AFM studies of refolding 21 were fully consistent with a two-state folding model , without misfolding ., More recent AFM 22 studies have suggested the formation of partially folded or misfolded species , which have been attributed to partial domain swapping in simulations 23 , but these are qualitatively different from the fully domain-swapped species considered here ., Therefore , it is interesting to ask the general questions: when included in tandem repeats , what types of protein structures are most likely to form domain-swapped misfolded states , and by what mechanism ?, In order to investigate the misfolding propensity of different types of domains , we have chosen seven domains , based on, ( i ) the superfamilies with the largest abundance of repeats in the human genome 24 ,, ( ii ) proteins for which some experimental evidence for misfolding ( or lack thereof ) is available and, ( iii ) proteins for which data on folding kinetics and stability is available for their circular permutants ( only some of the proteins meet criterion, ( iii ) ) ., The circular permutant data are relevant because the misfolding intermediates suggested by simulations and experiment 7 , 12 can be viewed as circular permutants of the original structure ( Fig 1d ) ., Each of the chosen proteins is illustrated in Fig 2 and described briefly in Materials and Methods ., We study the folding and misfolding of the seven protein domains , using the same structure-based model as that successfully employed to treat Titin I27 7 , 12 ., Molecular simulations are carried out to characterize the possible structural topologies of the misfolded intermediates and the mechanism of their formation ., Our model is consistent with available experimental information for the systems studied , in terms of which proteins misfold and what misfolded structures they tend to form ., We then investigated what factors influence the propensity of multidomain proteins to misfold ., The simplest rationalization of the propensity of a multidomain protein for domain-swapped misfolding would seem to be offered by parameterizing a kinetic model based on the scheme shown in Fig 1 , particularly for the steps Fig 1a–1b versus 1a–1d ., We hypothesized that the propensity to misfold might be characterized in terms of the folding kinetics of the isolated circular permutants representing the domain-swapped intermediates in Fig 1d ., However , contrary to this expectation , we found that the stability of such isolated domains , rather than their folding rate , is the main determinant of misfolding propensity ., Although superficially this appears to differ from previously suggested kinetic models 12 , it is completely consistent , with a specific interpretation of the rates ., Building on this understanding , we developed a very simplified model which can be used to predict which domains are likely to be susceptible to domain-swapped misfolding ., Finally , we have investigated the effect of the composition and length of the linker between the tandem repeats on the misfolding propensity ., Tandem Src homology 3 ( SH3 ) domains ( Fig 2a ) are widely found in signal transduction proteins and they share functions such as mediating protein-protein interactions and regulating ligand binding 25 ., Kinetic and thermodynamic properties of native and all the possible circular permutations of SH3 single domain have been well characterized 26 ., Two different circular permutant constructs of the sequence are known to fold to a circularly permuted native conformation ( PDB accession codes are 1TUC and 1TUD ) that is similar to the wild-tpe ( WT ) protein 26 ., With a similar function to the SH3 domains , Src homology 2 ( SH2 ) domains ( Fig 2b ) are also involved in the mediation of intra- and intermolecular interactions that are important in signal transduction 27 ., The SH2 domains are well-known from crystallographic analysis to form metastable domain-swapped dimers 28 , 29 ., Fibronectin type III ( fn3 ) domains ( Fig 2c ) are highly abundant in multidomain proteins , and often involved in cell adhesion ., We have chosen to study the third fn3 domain of human tenascin ( TNfn3 ) , which has been used as a model system to study the mechanical properties of this family ., Single-molecule AFM experiments revealed that a small fraction ( ∼ 4% ) of domains in native tenascin ( i . e . the full tenascin protein containing both TNfn3 and other fn3 domains ) 8 , with a similar signature to that observed for I27 ., Subsequently , misfolding events have been identified in a polyprotein consisting of repeats of TNfn3 only 9 ., Interestingly , a structure has been determined for a domain-swapped dimer of TNfn3 involving a small change of the loop between the second and third strand 30 ., PDZ domains ( Fig 2d ) are one of the most common modular protein-interaction domains 31 , recognizing specific-sequence motifs that occur at the C-terminus of target proteins or internal motifs that mimic the C-terminus structurally 32 ., Naturally occurring circularly permuted PDZ domains have been well studied 33–35 , and domain-swapped dimers of PDZ domains have been characterized by NMR spectroscopy 36 , 37 ., Titin ( Fig 2e ) is a giant protein spanning the entire muscle sarcomere 38 ., The majority of titin’s I-band region functions as a molecular spring which maintains the structural arrangement and extensibility of muscle filaments 39 ., The misfolding and aggregation properties of selected tandem Ig-like domains from the I-band of human Titin ( I27 , I28 and I32 ) have been extensively studied by FRET experiments 7 , 24 ., In the earlier work on tandem repeats of I27 domains , around 2% misfolding events were reported in repeated stretch-release cycles in AFM experiments 8 ., A slightly larger fraction ( ∼ 6% ) of misfolded species was identified in single-molecule FRET experiments and rationalized in terms of domain swapped intermediates , captured by coarse-grained simulations 7 , 11 ., In contrast , with the above misfolding-prone systems , there are certain polyprotein chains have been shown be resistant to misfolding , according to pulling experiments ., For instance little evidence for misfolding was identified in a polyprotein of GB1 14 ( Fig 2g ) , with more than 99 . 8% of the chains ( GB1 ) 8 folding correctly in repetitive stretching–relaxation cycles 14 ., Lastly , we consider polyUbiquitin ( Fig 2f ) , for which there is conflicting experimental evidence on misfolding ., Initial force microscopy studies showed only the formation of native folds 15 , with no misfolding ., Later work suggested the formation of collapsed intermediates 22 , however the signature change in molecular extension of these was different from that expected for fully domain-swapped misfolds ., A separate study using a lock-in AFM 21 found Ubiquitin to conform closely to expectations for a two-state folder , without evidence of misfolding ., For this protein , there is a strong imperative to avoid misfolding , since Ubiquitin is initially expressed as a tandem polyUbiquitin chain in which adjacent domains have 100% sequence identity , yet this molecule is critical for maintaining cellular homeostasis 40 ., A coarse grained structure-based ( Go-like ) model similar to the earlier work is employed for the study here 7 , 41 ., Each residue is represented by one bead , native interactions are attractive and the relative contact energies are set according to the Miyazawa–Jernigan matrix ., The model is based on that described by Karanicolas and Brooks 41 , but with native-like interactions allowed to occur between domains as well as within the same domain , as described below 7 ., All the simulations are run under a modified version of GROMACS 42 ., For the seven species we studied in this work , the native structures of single domains that were used to construct the models for SH3 , SH2 , PDZ , TNfn3 , Titin I27 , GB1 and Ubiquitin correspond to PDB entries 1SHG 43 , 1TZE 44 , 2VWR , 1TEN 45 , 1TIT 46 , 1GB1 47 and 1UBQ 48 respectively ., For the single domains of SH3 ( 1SHG ) , TNfn3 ( 1TEN ) and GB1 ( 1GB1 ) , additional linker sequences of Asp-Glu-Thr-Gly , Gly-Leu and Arg-Ser , respectively , are added between the two domains to mimic the constructs used in the corresponding experiments 9 , 14 , 26 ., Construction of the Titin I27 model was described in our previous work 7 ., In order to allow for domain-swapped misfolding , the native contact potentials within a single domain are also allowed to occur between corresponding residues in different domains , with equal strength ., Specifically , considering each single repeat of the dimeric tandem that has L amino acids , given any pair of residues ( with indices i and j ) that are the native interactions within a single domain , the interaction energy for the intradomain interaction ( Ei , j ( r ) ) is the same as the interdomain interaction between the residue ( i or j ) and the corresponding residue ( j + L or i + L ) in the adjacent domain , i . e . Ei , j ( r ) = Ei+L , j ( r ) = Ei , j+L ( r ) = Ei+L , j+L ( r ) ., To investigate the folding kinetics of the dimeric tandem , a total of 1024 independent simulations are performed on each system for a duration of 12 microseconds each ., Different misfolding propensities are observed at the end of the simulations ., With the exception of Ubiquitin and GB1 , the vast majority of the simulations reached stable native states with separately folded domains ., A small fraction of simulations form stable domain-swapped misfolded states ., All the simulations are started from a fully extended structure , and run using Langevin dynamics with a friction of 0 . 1 ps−1 and a time step of 10 fs ., We note that all the generated domain-swapped misfolding structures , containing the central and terminal domains , can be monitored by a reaction coordinate based on circular permutated native-like contact sets ., Each circularly permuted misfold can be characterized according to the loop position K in sequence where the native domain would be cut to form the circular permutant ( K = 0 corresponds to the native fold ) ., If a native contact Cnative = ( i , j ) exists between residues i and j in the native fold , the corresponding native-like contacts for the central ( Cin ( K ) ) and terminal domains ( Cout ( K ) ) of the domain swapped conformation are generated as, C i n ( K ) = ( i + Θ ( K − i ) L , j + Θ ( K − j ) L ) , C o u t ( K ) = ( i + Θ ( i − K ) L , j + Θ ( j − K ) L ) ,, where Θ ( x ) is the Heaviside step function and L is the length of each single domain ( plus interdomain linker ) ., Sin , K is the set of native-like contacts Cin of the central domain , and Sout , K is the set of all the native-like contacts Cout of the terminal domain ., Sin , K and Sout , K can be used to define a contact-based reaction coordinate to analyze the kinetics of the dimeric tandem misfolding ., The corresponding fraction of contacts for the central domain could be calculated by:, QK ( χ ) =1N∑ ( i , j ) ∈Sin , K11+eβ ( rij ( χ ) −λrij0 ) , ( 1 ), where N is the total number of domain swapped contacts , SK = Sin , K ∪ Sout , K ( equal to the total number of native contacts ) , rij ( χ ) is the distance between residue i and j in the protein configuration χ ., r i j 0 is the corresponding distance in the native structure for native-like contacts , β = 50 nm−1 and λ = 1 . 2 is used to account for fluctuations about the native contact distance ., The equilibrium properties of a single domain of each system are obtained from umbrella sampling along the native contacts Q as the reaction coordinate ., The obtained melting temperature of each system is listed in Table A in S1 Text ., A temperature at which the folding barrier ΔGf of approximately ∼ 2 . 5 kBT is chosen for the 2-domain tandem simulations for reasons described below ., The stability ΔGs is calculated as, Δ G s = - k B T ln ∫ Q ‡ 1 e - F ( Q ) / k B T d Q / ∫ 0 Q ‡ e - F ( Q ) / k B T d Q , ( 2 ), where kB and T are the Boltzmann constant and temperature respectively ., Q‡ is the position of the barrier top in F ( Q ) , separating the folded and unfolded states and F ( Q ) represents the free energy profile on Q . Barrier heights ΔGf were simply defined as ΔGf = G ( Q‡ ) − G ( Qu ) , where Qu is the position of the unfolded state free energy minimum on Q . We calculated the relative contact order 49 , RCOK of different circular permutants K via, RCO K = 1 L · N ∑ ( i , j ) ∈ S in , K | i - j | , ( 3 ), where L is the length of the single domain , and N is the total number of the native like contacts ( the same for different K ) ., Sin , K is the contacts set of the circular permutant corresponding to the “central domain” of the misfolded state ., Note that the contact order calculation here is using residue-based native contacts ( the same ones defined as attractive in the Gō model ) , instead of all atom native contacts ., An Ising-like model was built based on the native contact map , in which each residue is considered either folded or unfolded and so any individual configuration can be specified as a binary sequence , in a similar spirit to earlier work 50–52 ., Interactions between residues separated by more than two residues in the sequence are considered ., To simplify the analysis , we also consider that native structure grows only in a single stretch of contiguous native residues ( native segment ) , which means the configurations such as …UFFFUUUUU… or …UUUUUFFFU… are allowed , however , …UFFFUUUFFFU… is not allowed ( “single sequence approximation” ) 50 ., Each residue which becomes native incurs an entropy penalty ΔS , while all possible native contacts involving residues within the native segment are considered to be formed , each with a favourable energy of contact formation ϵ ., The partition function for such a model can be enumerated as:, Z = ∑ χ exp  − G ( χ ) k B T  = ∑ χ exp  − n ( χ ) ϵ − N f ( χ ) T Δ s k B T , where kB and T are the Boltzmann constant and temperature ., G ( χ ) is the free energy determined by the number of native contacts n ( χ ) in the configuration χ , and the number of native residues , Nf ( χ ) ., The distribution of the microstates ( χ ) can be efficiently generated by the Metropolis-Hastings method with Monte Carlo simulation ., In each iteration , the state of one randomly chosen residue ( among the residues at the two ends of the native fragment and their two neighbouring residues ) is perturbed by a flip , from native to unfolded or from unfolded to native , taking the system from a microstate χ1 with energy E1 to a microstate χ2 with energy E2 ., The new microstate is subject to an accept/reject step with acceptance probability, P acc = min  1 , exp ( - E 2 - E 1 k B T )  ., ( 4 ) To mimic the folding stability difference between native and circular permutant folds , a penalty energy term Ep has been added whenever the native fragment crosses the midpoint of the sequence from either side ( the function θ ( χ ) above is 1 if this is true , otherwise zero ) ., That situation corresponds to formation of a domain-swapped structure , in which there is additional strain energy from linking the termini , represented by Ep ., We only use the Ising model here to investigate formation of the first domain ( either native or circular permutant ) , by rejecting any proposed Monte Carlo step that would make the native segment longer than the length of single domain , L ., In order to characterize the potential misfolding properties of each type of domain , we have used a Gō-type energy function based on the native structure ., Such models have successfully captured many aspects of protein folding , including ϕ-values 53 , 54 , dimerization mechanism 55 , 56 , domain-swapping 57–60 , and the response of proteins to a pulling force 61 , 62 ., More specifically , a Gō type model was used in conjunction with single-molecule and ensemble FRET data to characterize the misfolded states and misfolding mechanism of engineered tandem repeats of Titin I27 7 , 12 ., We have therefore adopted the same model ., Although it is based on native-contacts , it can describe the type of misfolding we consider here , which is also based on native-like structure ., Note that this model effectively assumes 100% sequence identity between adjacent domains , the scenario that would most likely lead to domain-swap formation ., It is nonetheless a relevant limit for this study , as there are examples in our data set of adjacent domains having identical sequences which do misfold ( e . g . titin I27 ) and those which do not ( e . g . protein G ) ., For each of the folds shown in Fig 2 , we ran a large number of simulations , starting from a fully extended , unfolded chain , for sufficiently long ( 12 μs each ) such that the vast majority of them reached either the correctly folded tandem dimer , or a domain-swapped misfolded state similar to that shown in Fig 1e for titin ., In fact , for each protein , a number of different misfolded topologies are possible , illustrated for the Src SH3 domain in Fig 3 ., Each of these domains , shown in conventional three-dimensional cartoon representation in the right column of Fig 3 and in a simplified two-dimensional topology map in the left column , consists of two native-like folded ( or misfolded ) domains ., For convenience , we call the domain formed from the central portion of the sequence the “central domain” and that from the terminal portions the “terminal domain” ., We have chosen to characterize each topology in terms of the position , K , in sequence after which the central domain begins ., Thus , the native fold has K = 0 , and all the misfolded states have K > 0 . Typically , because of the nature of domain swapping , K must fall within a loop ., Of course , there is a range of residues within the loop in question that could be identified as K and we have merely chosen a single K close to the centre of the loop ., This position , and the central domain , are indicated for the Src SH3 misfolded structures in Fig 3 ., We note that each of these central domains can also be considered as a circular permutant of the native fold , in which the ends of the protein have been joined and the chain has been cut at position K . With this nomenclature in hand , we can more easily describe the outcome of the folding simulations for the seven domain types considered in terms of the fraction of the final frames that belonged to the native fold , versus each of the possible misfolded states ., These final populations are shown in Table 1 ., We see that for five of the domains ( SH3 , SH2 , PDZ , TNfn3 , Titin I27 ) , misfolded structures are observed , with total populations ranging from 5–10% ., For the remaining two domains , Ubiquitin ( UBQ ) and protein G ( GB1 ) , no misfolded population is observed ., The ability to capture domain-swapped misfolds with simple coarse-grained simulations potentially allows us to investigate the origin of the misfolding , and its relation , if any , to the topology of the domain in question ., However , we also need to benchmark the accuracy of the results against experiment as far as possible , in order to show that they are relevant ., There are two main sources of information to validate our results ., The first is the overall degree of domain-swapped misfolding for those proteins where it has been characterized , for example by single molecule AFM or FRET experiments ., Qualitatively we do observe good agreement , where data is available: in experiment , domains which have been shown to misfold are TNfn3 ( AFM ) and Titin I27 ( AFM , FRET ) , which are both found to misfold here , while there is no detectable misfolded population for protein G ( AFM ) , again consistent with our results ., We also do not observe any misfolding for Ubiquitin , consistent with the lack of experimental evidence for fully domain-swapped species for this protein 15–23 ., Quantitatively , the fractional misfolded population is also consistent with the available experimental data ., For instance , the frequency of misfolded domains in native tenascin is ∼ 4% as shown by previous AFM experiments 8 , the misfolded population of I27 dimers is ∼5% in single-molecule FRET experiments 7 while the misfolded population of GB1 domains in polyproteins ( GB18 ) is extrememly low ( < 0 . 2% ) 14 ., Even though the observed misfolding population of the misfolded tandem dimer is low , it is potentially a problem considering that many of the multidomain proteins in nature have large number of tandem repeats , such as Titin which contains twenty-two I27 repeats 63 ., Recent FRET experiments on I27 tandem repeats have shown that the fraction of misfolded proteins increases with the number of repeats ., For the 3- and 8-domain polyproteins , the fraction of misfolded domains increases by a factor of 1 . 3 and 1 . 8 , respectively , relative to a tandem dimer 12 ., The second type of evidence comes from experimental structures of domain-swapped dimers ., For several of the proteins , bimolecular domain-swapped structures have been determined experimentally ., While no such structures have yet been determined for single-chain tandem dimers , we can compare the misfolded states with the available experimental data ., For each experimental example , we are able to find a corresponding misfolded species in our simulation with very similar structure ( related by joining the terminis of the two chains in the experimental structures ) ., The domain swapped dimers solved obtained from experiments ( Fig 4a , 4c , 4e and 4g ) are strikingly similar to the domain swapping dimeric tandem from simulations , which are the domain swapped SH3 domains when K ( sequence position after which the central domain begins ) = 37 ( Fig 4b ) , SH2 with K = 72 ( Fig 4d ) , TNfn3 with K = 28 ( Fig 4f ) and PDZ with K = 23 ( Fig 4h ) ., Most of these states have relatively high population among all the possible misfolds as observed from the simulations ( “Population” in Table 1 ) ., While the coverage of possible domain swaps is by no means exhaustive , the observed correspondence gives us confidence that the misfolded states in the simulations are physically plausible ., Having shown that the misfolding propensities we obtain are qualitatively consistent with experimental evidence ( and in the case of Titin I27 , in semi-quantitative agreement with single-molecule FRET ) , we set out to establish some general principles relating the properties of each domain to its propensity to misfold in this way ., We can start to formulate a hypothesis based on the alternative folding and misfolding pathways illustrated in Fig 1 ., Native folding has as an intermediate a state in which either the N- or the C-terminal domain is folded ., In contrast , on the misfolding pathway , the first step is formation of the central domain , followed by that of the terminal domain ., This parallel pathway scheme suggests that a descriptor of the overall misfolding propensity may be obtained from the rate of formation of a single correctly folded domain , relative to that of the central domain ( neglecting back reactions , because this are rarely seen in our simulations ) ., We can study the central domain formation in isolation , since these structures are just circular permutants of the native fold , i . e . the two proteins have the same sequence as the native , but with the position of the protein termini moved to a different point in the sequence , as is also found in nature 35 ., These structures can be thought of as originating from the native by cutting a specific loop connecting secondary structure elements ( the free energy cost of splitting such an element being too high ) , and splicing together the N- and C- termini ., In the context of the tandem dimers , the position at which the loop is cut is the same K that defines the start of the central domain in sequence ., We investigate the role of the central domain by characterizing the free energy landscape of the single domain of each system , as well as all of its possible circular permutants , using umbrella sampling along the reaction coordinate QK ., QK is exactly analogous to the conventional fraction of native contacts coordinate Q 64 , but defined using the corresponding ( frame-shifted ) contacts in the circular permutant pseudo-native structure ., The index K indicates the position along the sequence of the WT where the cut is made in order to convert to the circular permutant ., The free energy surfaces F ( QK ) of two representative systems , SH3 and Ubiquitin , are shown in Fig 5 , with the data for the remaining proteins given in the Fig A in S1 Text ., The free energy barrier height for folding ΔGf and the stability ΔGs are listed in the Table 1 ., The free energy plots indicate that the single domains of Ubiquitin and GB1 are stable only for the native sequence order , and not for any of the circular permutants ., Based on the type of misfolding mechanism sketched in Fig 1 , one would expect that unstable circular permutants would result in an unstable central domain , and consequently no stable domain-swappping misfolding would occur in the dimer folding simulations , as we indeed observe ., This is also consistent with previous studies of polyproteins of GB1 and Ubiquitin using using AFM experiments , which reveal high-fidelity folding and refolding 14 , 65 , 66 ., We note that only under very strongly stabilizing conditions is any misfolding observed for ubiquitin dimers: running simulations at a lower temperature ( 260 K ) , we observe a very small ( 1 . 3% ) population of misfolded states from 1024 trial folding simulations ., At a higher temperature of 295 K , once again no misfolding is observed ., In contrast to the situation for GB1 and Ubiquitin , all of the circular permutants of the SH3 domain in Fig 5 are in fact stable , although less so than the native fold ., The destabilization of circular permutants relative to native is in accord with the experimental results for the Src SH3 domain 26 ( rank correlation coefficient stabilities is 0 . 80 ) ., The other domains considered also have stable circular permutant structures ., This is consistent with the fact that all of these domains do in fact form some fraction of domain-swapped misfolded states ., The simplest view of the misfolding mechanism would be as a kinetic competition between the correctly folded intermediates versus the domain-swapped intermediates with a central domain folded ( i . e . a “kinetic partitioning” mechanism 67 ) ., In this case one might naively expect that the propensity to misfold would be correlated with the relative folding rates of an isolated native domain and an isolated circular permutant structure ., However , the folding barriers ΔGf projected onto Q ( for native ) or QK ( for circular permutants ) show little correlation to the relative frequency of the corresponding folded or misfolded state , when considering all proteins ( Table 1 ) ., Since this barrier height may not reflect variations in the folding rate if some of the coordinates are poor ( yielding a low barrier ) or if there are large differences in kinetic prefactors , we have also directly computed the folding rate for the circular permutants of those proteins which misfold , and confirm that the rates of formation of the native fold and circular permutants are similar ., We indeed obtain a strong correlation between the folding rate o
Recent single molecule experiments , using either atomic force microscopy ( AFM ) or Förster resonance energy transfer ( FRET ) have shown that multidomain proteins containing tandem repeats may form stable misfolded structures ., Topology-based simulation models have been used successfully to generate models for these structures with domain-swapped features , fully consistent with the available data ., However , it is also known that some multidomain protein folds exhibit no evidence for misfolding , even when adjacent domains have identical sequences ., Here we pose the question: what factors influence the propensity of a given fold to undergo domain-swapped misfolding ?, Using a coarse-grained simulation model , we can reproduce the known propensities of multidomain proteins to form domain-swapped misfolds , where data is available ., Contrary to what might be naively expected based on the previously described misfolding mechanism , we find that the extent of misfolding is not determined by the relative folding rates or barrier heights for forming the domains present in the initial intermediates leading to folded or misfolded structures ., Instead , it appears that the propensity is more closely related to the relative stability of the domains present in folded and misfolded intermediates ., We show that these findings can be rationalized if the folded and misfolded domains are part of the same folding funnel , with commitment to one structure or the other occurring only at a relatively late stage of folding ., Nonetheless , the results are still fully consistent with the kinetic models previously proposed to explain misfolding , with a specific interpretation of the observed rate coefficients ., Finally , we investigate the relation between interdomain linker length and misfolding , and propose a simple alchemical model to predict the propensity for domain-swapped misfolding of multidomain proteins .
Multidomain proteins with tandem repeats are abundant in eukaryotic proteins ., Recent studies have shown that such domains may have a propensity for forming domain-swapped misfolded species which are stable for long periods , and therefore a potential hazard in the cell ., However , for some types of tandem domains , no detectable misfolding was observed ., In this work , we use coarse-grained structure-based folding models to address two central questions regarding misfolding of multidomain proteins ., First , what are the possible structural topologies of the misfolds for a given domain , and what determines their relative abundance ?, Second , what is the effect of the topology of the domains on their propensity for misfolding ?, We show how the propensity of a given domain to misfold can be correlated with the stability of domains present in the intermediates on the folding and misfolding pathways , consistent with the energy landscape view of protein folding ., Based on these observations , we propose a simplified model that can be used to predict misfolding propensity for other multidomain proteins .
simulation and modeling, fluorophotometry, protein structure, thermodynamics, research and analysis methods, fluorescence resonance energy transfer, proteins, structural proteins, repeated sequences, molecular biology, spectrophotometry, free energy, physics, biochemistry, biochemical simulations, tandem repeats, protein domains, genetics, biology and life sciences, physical sciences, genomics, computational biology, spectrum analysis techniques, macromolecular structure analysis