C‐terminal deletion of leucine‐rich repeats from YopM reveals a heterogeneous distribution of stability in a cooperatively folded protein

Terminal deletions of units from α‐helical repeat proteins have provided insight into the physical origins of their cooperativity. To test if the same principles governing cooperativity apply to β‐sheet‐containing repeat proteins, we have created a series of C‐terminal deletion constructs from a large leucine‐rich repeat (LRR) protein, YopM. We have examined the structure and stability of the resulting deletion constructs by a combination of solution spectroscopy, equilibrium denaturation studies, and limited proteolysis. Surprisingly, a high degree of nonuniformity was found in the stability distribution of YopM. Unlike previously studied repeat proteins, we identified several key LRR that on deletion disrupt nearby structure, at distances as far away as up to three repeats, in YopM. This partial unfolding model is supported by limited proteolysis studies and by point substitution in repeats predicted to be disordered as a result of deletion of adjacent repeats. We show that key internal‐ and terminal‐caps must be present to maintain the structural integrity in adjacent regions (roughly four LRRs long) of decreased stability. The finding that full‐length YopM maintains a high level of cooperativity in equilibrium unfolding underscores the importance of interfacial interactions in stabilizing locally unstable regions of structure.     

The tolerance of a modular protein to duplication and deletion of internal repeats

Ankyrin repeat polypeptides contain repeated structural elements that pack to produce modular architectures lacking in close contacts between distant segments of the polypeptide chain. Despite this lack of sequence-distant contacts, ankyrin repeat polypeptides have been shown to fold in a cooperative manner. To determine the distance over which cooperative interactions can be propagated in a repeat protein, and to investigate the tolerance to internal duplication and deletion of modules, we have constructed a series of ankyrin repeat variants of the Notch ankyrin domain in which repeat number is varied by duplication and deletion of internal repeats. A construct with two copies of the fifth ankyrin repeat shows a modest increase in stability compared to the parent construct and retains apparent two-state unfolding behavior. Although constructs containing three and four copies of the fifth repeat retain this increased resistance to urea, they exhibit broad, multi-state unfolding transitions compared to the parent construct. For the Notch ankyrin domain, these larger constructs may represent a limit beyond which full cooperativity cannot be maintained. Deletions of internal repeats from the Notch ankyrin domain significantly destabilize the domain. This severe destabilization, which is larger than that resulting from end-repeat deletion, may arise from unfavorable interactions within the new non-native interfaces produced by internal repeat deletion. These results demonstrate both an asymmetry between the duplication and deletion of internal repeats, and a difference between deletion of internal and end-repeats, suggesting preferred mechanisms for evolution of repeat proteins.     

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-?N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1–3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action.     

Crystal structure and standardized geometric analysis of InlJ, a listerial virulence factor and leucine-rich repeat protein with a novel cysteine ladder

We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-Å resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain. InlJ bears 15 leucine-rich repeats (LRRs), the same number as in InlA, the prototypical internalin family member. The LRRs of InlJ differ from those of other internalins by having 21, rather than 22, residues and by replacing 1 LRR-defining hydrophobic residue with a conserved cysteine. These cysteines stack to form an intramolecular ladder and regular hydrophobic interactions in consecutive repeats. Analyzing the curvature, twist, and lateral bending angles of InlJ and comparing these with several other LRR proteins, we provide a systematic geometric comparison of LRR protein structures (http://bragi2.helmholtz-hzi.de/Angulator/). These indicate that both cysteine and asparagine ladders stabilize the LRR fold, whereas substitutions in some repeat positions are more likely than others to induce changes in LRR geometry.     

Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspects of protein design and engineering. Previously, we designed a series of consensus ankyrin repeat proteins containing one, two, three and four identical repeats (1ANK, 2ANK, 3ANK and 4ANK). These proteins, particularly 4ANK, are intended for use as a universal scaffold on which specific binding sites can be constructed. Despite being well folded and extremely stable, 4ANK is soluble only under acidic conditions. Designing interactions with naturally occurring proteins requires the designed protein to be soluble at physiological pH. Substitution of six leucines with arginine on exposed hydrophobic patches on the surface of 4ANK resulted in increased solubility over a large pH range. Study of the pH dependence of stability demonstrated that 4ANK is one of the most stable ankyrin repeat proteins known. In addition, analogous leucine to arginine substitutions on the surface of 2ANK allowed the partially folded protein to assume a fully folded conformation. Our studies indicate that replacement of surface-exposed hydrophobic residues with positively charged residues can significantly improve protein solubility at physiological pH.     

LRDD, a novel leucine rich repeat and death domain containing protein

Death domains (DD) and leucine rich repeats (LRR) are two different types of protein interaction motifs. Death domains are found predominantly in proteins involved in signaling and are involved in homo- and heteromultimerization. Leucine rich repeats are found in proteins with diverse cellular functions, like cell adhesion and cellular signaling, and mediate reversible protein-protein interactions. In this paper we report the cloning of a new human gene called LRDD (leucine repeat death domain containing protein). LRDD encodes a protein of 83 kDa with six LRRs at the N-terminus and a DD at the C-terminus. LRDD appears to be processed into two fragments of about 33 and 55 kDa, containing LRRs and DD respectively. Interestingly, LRDD is shown to interact with two other death domain containing proteins, FADD and MADD, presumably through death domain interactions. LRDD may represent a new type of adapter protein that could be involved in signaling or other cellular functions.     

Protein aggregation determinants from a simplified model: cooperative folders resist aggregation

Two-chain aggregation simulations using minimalist models of proteins G, L, and mutants were used to investigate the fundamentals of protein aggregation. Mutations were selected to break up repeats of hydrophobic beads in the sequence while maintaining native topology and folding ability. Data are collected under conditions in which all chain types have similar folded populations and after equilibrating the separated chains to minimize competition between folding and aggregation. Folding cooperativity stands out as the best single-chain determinant under these conditions and for these simple models. It can be experimentally measured by the width of the unfolding transition during thermal denaturation and loosely related to population of intermediate-like states during folding. Additional measures of cooperativity and other properties such as radius of gyration fluctuations and patterning of hydrophobic residues are also examined. Initial contact system states with transition-state characteristics can be identified and are more expanded than average initial contact states. Two-chain minimalist model aggregates are considerably less structured than their native states and have minimal domain-swapping features.     

Modulation of Folding Kinetics of Repeat Proteins: Interplay between Intra- and Interdomain Interactions

Repeat proteins have unique elongated structures that, unlike globular proteins, are quite modular. Despite their simple one-dimensional structure, repeat proteins exhibit intricate folding behavior with a complexity similar to that of globular proteins. Therefore, repeat proteins allow one to quantify fundamental aspects of the biophysics of protein folding. One important feature of repeat proteins is the interfaces between the repeating units. In particular, the distribution of stabilities within and between the repeats was previously suggested to affect their folding characteristics. In this study, we explore how the interface affects folding kinetics and cooperativity by investigating two families of repeat proteins, namely, the Ankyrin and tetratricopeptide repeat proteins, which differ in the number of interfacial contacts that are formed between their units as well as in their folding behavior. By using simple topology-based models, we show that modulating the energetic strength of the interface relative to that of the repeat itself can drastically change the protein stability, folding rate, and cooperativity. By further dissecting the interfacial contacts into several subsets, we isolated the effects of each of these groups on folding kinetics. Our study highlights the importance of interface connectivity in determining the folding behavior.     

Enhancing the stability and folding rate of a repeat protein through the addition of consensus repeats

Repeat proteins are constructed from a linear array of modular units, giving rise to an overall topology lacking long-range interactions. This suggests that stabilizing repeat modules based on consensus information might be added to a repeat protein domain, allowing it to be extended without altering its overall topology. Here we add consensus modules the ankyrin repeat domain from the Drosophila Notch receptor to investigate the structural tolerance to these modules, the relative thermodynamic stability of these hybrid proteins, and how alterations in the energy landscape influence folding kinetics. Insertions of consensus modules between repeats five and six of the Notch ankyrin domain have little effect on the far and near-UV CD spectra, indicating that neither secondary nor tertiary structure is dramatically altered. Furthermore, stable structure is maintained at increased denaturant concentrations in the polypeptides containing the consensus repeats, indicating that the consensus modules are capable of stabilizing much of the domain. However, insertion of the consensus repeats appears to disrupt cooperativity, producing a two-stage (three-state) unfolding transition in which the C-terminal repeats unfold at moderate urea concentrations. Removing the C-terminal repeats (Notch ankyrin repeats six and seven) restores equilibrium two-state folding and demonstrates that the high stability of the consensus repeats is propagated into the N-terminal, naturally occurring Notch ankyrin repeats. This stability increase greatly increases the folding rate, and suggests that the transition state ensemble may be repositioned in the chimeric consensus-stabilized proteins in response to local stability.     

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.     

Capping motifs stabilize the leucine‐rich repeat protein PP32 and rigidify adjacent repeats

Capping motifs are found to flank most β‐strand‐containing repeat proteins. To better understand the roles of these capping motifs in organizing structure and stability, we carried out folding and solution NMR studies on the leucine‐rich repeat (LRR) domain of PP32, which is composed of five tandem LRR, capped by α‐helical and β‐hairpin motifs on the N‐ and C‐termini. We were able to purify PP32 constructs lacking either cap and containing destabilizing substitutions. Removing the C‐cap results in complete unfolding of PP32. Removing the N‐cap has a much less severe effect, decreasing stability but retaining much of its secondary structure. In contrast, the dynamics and tertiary structure of the first two repeats are significantly perturbed, based on ¹H‐¹⁵N relaxation studies, chemical shift perturbations, and residual dipolar couplings. However, more distal repeats (3 to C‐cap) retain their native tertiary structure. In this regard, the N‐cap drives the folding of adjacent repeats from what appears to be a molten‐globule‐like state. This interpretation is supported by extensive analysis using core packing substitutions in the full‐length and N‐cap‐truncated PP32. This work highlights the importance of caps to the stability and structural integrity of β‐strand‐containing LRR proteins, and emphasizes the different contributions of the N‐ and C‐terminal caps.     

The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain

Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35‐residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic–aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D‐NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair‐wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic–aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.     

A new approach to the design of uniquely folded thermally stable proteins

A new computer program (CORE) is described that predicts core hydrophobic sequences of predetermined target protein structures. A novel scoring function is employed, which for the first time incorporates parameters directly correlated to free energies of unfolding (deltaGu), melting temperatures (Tm), and cooperativity. Metropolis-driven simulated annealing and low-temperature Monte Carlo sampling are used to optimize this score, generating sequences predicted to yield uniquely folded, stable proteins with cooperative unfolding transitions. The hydrophobic core residues of four natural proteins were predicted using CORE with the backbone structure and solvent exposed residues as input. In the two smaller proteins tested (Gbeta1, 11 core amino acids; 434 cro, 10 core amino acids), the native sequence was regenerated as well as the sequence of known thermally stable variants that exhibit cooperative denaturation transitions. Previously designed sequences of variants with lower thermal stability and weaker cooperativity were not predicted. In the two larger proteins tested (myoglobin, 32 core amino acids; methionine aminopeptidase, 63 core amino acids), sequences with corresponding side-chain conformations remarkably similar to that of native were predicted.     

A capping domain for LRR protein interaction modules.

Leucine-rich repeats (LRR) are protein interaction modules which are present in a large number of proteins with diverse functions. We describe here a novel motif (16-19 residues) downstream of the last, incomplete, LRR in a subfamily of LRR proteins. In the U2A' spliceosomal protein, this motif is folded into a cap that shields the hydrophobic core of the LRRs from the solvent. Modelling of the LRR-cap in the imidazoline-1 candidate receptor, using the known structure of U2A' as template, showed a conservation of the basic structural features.     

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4‐helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t‐butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.     

Conformational energy analysis of the leucine repeat regions of C/EBP,GCN4, and the proteins of themyc,jun, andfos oncogenes

It has been recently proposed that certain DNA binding proteins (including C/EBP, GCN4 and themyc, jun, andfos oncogene proteins) share a common structural motif based on helix-promoting regions containing heptad repeat sequences of leucines. It has been suggested that this structure is critical to the biological activity of these proteins, since it facilitates the formation of functional dimers held together by interdigitating leucine side-chains along the hydrophobic interfaces between long α-helical regions of the polypeptide chains in a configuration termed the “leucine zipper.” In this paper, conformational energy analysis is used to determine the preferred three-dimensional structures of the leucine repeat regions of these proteins. The results indicate that, in all cases, the global minimum energy conformation for these regions is an amphipathic α-helix with the leucine side-chains arrayed on one side in such a way to favor “leucine zipper” dimerization. Furthermore, amino acid substitutions in these regions (such as Pro for Leu), that are known to inhibit dimer formation and prevent DNA binding, are found to produce significant conformational changes that disrupt the amphipathic helical structure. Thus, these results provide support for the proposed “leucine zipper” configuration as a critical structural feature of this class of DNA binding proteins.

     

Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins

Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins (∼ 400-800 s^− 1), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10⁴ with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.     

Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded alpha-helical coiled-coils

Detailed sequence analyses of the hydrophobic core residues of two long two-stranded alpha-helical coiled-coils that differ dramatically in sequence, function, and length were performed (tropomyosin of 284 residues and the coiled-coil domain of the myosin rod of 1086 residues). Three types of regions were present in the hydrophobic core of both proteins: stabilizing clusters and destabilizing clusters, defined as three or more consecutive core residues of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (Gly, Ala, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Arg, Lys, and Trp) residues, and intervening regions that consist of both stabilizing and destabilizing residues in the hydrophobic core but no clusters. Subsequently, we designed a series of two-stranded coiled-coils to determine what defines a destabilizing cluster and varied the length of the destabilizing cluster from 3 to 7 residues to determine the length effect of the destabilizing cluster on protein stability. The results showed a dramatic destabilization, caused by a single Leu to Ala substitution, on formation of a 3-residue destabilizing cluster (DeltaT(m) of 17-21 degrees C) regardless of the stability of the coiled-coil. Any further substitution of Leu to Ala that increased the size of the destabilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (DeltaT(m) of 1.4-2.8 degrees C). These results suggested that the contribution of Leu to protein stability is context-dependent on whether the hydrophobe is in a stabilizing cluster or its proximity to neighboring destabilizing and stabilizing clusters.     

Influence of 5-N-carboxamide modifications on the thermodynamic stability of oligonucleotides

We have recently shown that the incorporation of modified nucleotides such as 5-N-carboxamide-deoxyuridines into random nucleic acid libraries improves success rates in SELEX experiments and facilitates the identification of ligands with slow off-rates. Here we report the impact of these modifications on the thermodynamic stability of both duplexes and intramolecular ‘single-stranded’ structures. Within duplexes, large, hydrophobic naphthyl groups were destabilizing relative to the all natural DNA duplex, while the hydrophilic groups exhibited somewhat improved duplex stability. All of the significant changes in stability were driven by opposing contributions from the enthalpic and entropic terms. In contrast, both benzyl and naphthyl modifications stabilized intramolecular single-stranded structures relative to their natural DNA analogs, consistent with the notion that intramolecular folding allows formation of novel, stabilizing hydrophobic interactions. Imino proton NMR data provided evidence that elements of the folded structure form at temperatures well below the T_m, with a melting transition that is distinctly less cooperative when compared to duplex DNA. Although there are no data to suggest that the unmodified DNA sequences fold into structures similar to their modified analogs, this still represents clear evidence that these modifications impart thermodynamic stability to the folded structure not achievable with unmodified DNA.     

Surface hydrophobic groups, stability, and flip-flopping in lattice proteins

Two-dimensional lattice protein models were studied in two approximations of the conformational equilibrium to elucidate the role of surface hydrophobic groups in their stabilities. We demonstrate that stability of any compactly folded sequence is determined by its ability to "flip-flop" (refold) into alternative compact structures. The degree of stability required for folded sequences determines the average numbers of surface hydrophobic groups in stable lattice structures which are in good agreement with ratios of core to surface hydrophobic groups in real proteins. However, the average destabilization of the native structure per surface hydrophobic group is small (0-0.25 kcal/mol), often disagrees with the free energies derived from the ratios of core to surface hydrophobic groups in the same structures, and has a combinatorial entropic nature independent of the strength of structure stabilizing interactions. This suggests that the free energies derived from the core to surface ratios of hydrophobic groups in real proteins have little to do with folding thermodynamics. On average, sequences with highly stable native structures are the least hydrophobic. The results suggest that in designing novel stable proteins hydrophobic groups on the surface should be avoided to reduce the possibility of flip-flopping. The average stability of highly designable structures is never higher than that of some low designability structures, contrary to the accepted view. In the equilibrium approximation with alternative compact and partially unfolded structures, the requirement of high stability selects a unique 5 x 5 structure formed by only a few sequences, suggesting much stronger sequence selectivity than commonly thought.