Folding Of Repeat Proteins Research Articles

Evolution and folding of repeat proteins

Repeat proteins are made with tandem copies of similar amino acid stretches that fold into elongated architectures. These proteins constitute excellent model systems to investigate how evolution relates to structure, folding, and function. Here, we propose a scheme to map evolutionary information at the sequence level to a coarse-grained model for repeat-protein folding and use it to investigate the folding of thousands of repeat proteins. We model the energetics by a combination of an inverse Potts-model scheme with an explicit mechanistic model of duplications and deletions of repeats to calculate the evolutionary parameters of the system at the single-residue level. These parameters are used to inform an Ising-like model that allows for the generation of folding curves, apparent domain emergence, and occupation of intermediate states that are highly compatible with experimental data in specific case studies. We analyzed the folding of thousands of natural Ankyrin repeat proteins and found that a multiplicity of folding mechanisms are possible. Fully cooperative all-or-none transitions are obtained for arrays with enough sequence-similar elements and strong interactions between them, while noncooperative element-by-element intermittent folding arose if the elements are dissimilar and the interactions between them are energetically weak. Additionally, we characterized nucleation-propagation and multidomain folding mechanisms. We show that the global stability and cooperativity of the repeating arrays can be predicted from simple sequence scores.

Analysis of Tandem Repeat Protein Folding Using Nearest-Neighbor Models.

Cooperativity is a hallmark of protein folding, but the thermodynamic origins of cooperativity are difficult to quantify. Tandem repeat proteins provide a unique experimental system to quantify cooperativity due to their internal symmetry and their tolerance of deletion, extension, and in some cases fragmentation into single repeats. Analysis of repeat proteins of different lengths with nearest-neighbor Ising models provides values for repeat folding ([Formula: see text]) and inter-repeat coupling (ΔGi-1,i). In this article, we review the architecture of repeat proteins and classify them in terms of ΔGi and ΔGi-1,i; this classification scheme groups repeat proteins according to their degree of cooperativity. We then present various statistical thermodynamic models, based on the 1D-Ising model, for analysis of different classes of repeat proteins. We use these models to analyze data for highly and moderately cooperative and noncooperative repeat proteins and relate their fitted parameters to overall structural features.

Designed folding pathway of modular coiled-coil-based proteins

Natural proteins are characterised by a complex folding pathway defined uniquely for each fold. Designed coiled-coil protein origami (CCPO) cages are distinct from natural compact proteins, since their fold is prescribed by discrete long-range interactions between orthogonal pairwise-interacting coiled-coil (CC) modules within a single polypeptide chain. Here, we demonstrate that CCPO proteins fold in a stepwise sequential pathway. Molecular dynamics simulations and stopped-flow Förster resonance energy transfer (FRET) measurements reveal that CCPO folding is dominated by the effective intra-chain distance between CC modules in the primary sequence and subsequent folding intermediates, allowing identical CC modules to be employed for multiple cage edges and thus relaxing CCPO cage design requirements. The number of orthogonal modules required for constructing a CCPO tetrahedron can be reduced from six to as little as three different CC modules. The stepwise modular nature of the folding pathway offers insights into the folding of tandem repeat proteins and can be exploited for the design of modular protein structures based on a given set of orthogonal modules.

A collection of programs for one-dimensional Ising analysis of linear repeat proteins with point substitutions.

A collection of programs is presented to analyze the thermodynamics of folding of linear repeat proteins using a 1D Ising model to determine intrinsic folding and interfacial coupling free energies. Expressions for folding transitions are generated for a series of constructs with different repeat numbers and are globally fitted to transitions for these constructs. These programs are designed to analyze Ising parameters for capped homopolymeric consensus repeat constructs as well as heteropolymeric constructs that contain point substitutions, providing a rigorous framework for analysis of the effects of mutation on intrinsic and directional (i.e., N- vs. C-terminal) interfacial coupling free-energies. A bootstrap analysis is provided to estimate parameter uncertainty as well as correlations among fitted parameters. Rigorous statistical analysis is essential for interpreting fits using the complex models required for Ising analysis of repeat proteins, especially heteropolymeric repeat proteins. Programs described here are available at https://github.com/barricklab-at-jhu/Ising_programs.

Cavities in Context: Distinct Consequences of Packing Defects in a Repeat Protein Folding Landscape

Cavities in Context: Distinct Consequences of Packing Defects in a Repeat Protein Folding Landscape

Cotranslational Folding of a Pentarepeat β-Helix Protein

It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum—a homolog of the fluoroquinolone resistance protein MfpA—using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is ~35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.

Context-Dependent Energetics of Loop Extensions in a Family of Tandem-Repeat Proteins

Consensus-designed tetratricopeptide repeat proteins are highly stable, modular proteins that are strikingly amenable to rational engineering. They therefore have tremendous potential as building blocks for biomaterials and biomedicine. Here, we explore the possibility of extending the loops between repeats to enable further diversification, and we investigate how this modification affects stability and folding cooperativity. We find that extending a single loop by up to 25 residues does not disrupt the overall protein structure, but, strikingly, the effect on stability is highly context-dependent: in a two-repeat array, destabilization is relatively small and can be accounted for purely in entropic terms, whereas extending a loop in the middle of a large array is much more costly because of weakening of the interaction between the repeats. Our findings provide important and, to our knowledge, new insights that increase our understanding of the structure, folding, and function of natural repeat proteins and the design of artificial repeat proteins in biotechnology.

Allostery and molecular machines.

Machines undergo repeated energy (fuel)-driven cycling between various functional and structural states. Such cycling involves motions of the machine parts, which take place in a highly coordinated manner in time and space. This description applies not only to man-made machines but also to the molecular machines that mediate many of the key processes in all forms of life [1]. Examples include protein synthesis by the ribosome, DNA unwinding by helicases during replication, protein degradation by the proteasome and protein folding by chaperones. The coordinated movements of most biomolecular machines are achieved by allosteric regulation of ATP binding and hydrolysis. Consequently, a molecular level understanding of how the essential machines of life work requires invoking and further developing allosteric theory. Hence, the motivation for the Royal Society Discussion Meeting entitled ‘Allostery and molecular machines' that took place in June 2017. The meeting brought together scientists interested in allostery, in general, with others who are studying specific biomolecular machines of interest. The meeting led to many interesting discussions and resulted in this special issue of the Philosophical Transactions B . In 1965, Monod, Wyman & Changeux published a seminal paper [2] that described a so-called ‘concerted model' (referred to here and generally as the MWC model) for cooperative ligand binding by oligomeric proteins. This issue contains a paper by one of the co-authors of this landmark paper in which the MWC model is explained in brief and its application to the nicotinic acetylcholine receptor is then described [3]. The MWC model remains important but its elegance has come at the price of several restrictive assumptions. One key assumption is that cooperativity is due to a shift in equilibrium between different quaternary …

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1H-15N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.

The Effect of Loop Insertions on the Folding of Tandem-Repeat Proteins

Multi-sequence alignment and the identification of conserved residues have allowed the design of highly stable consensus repeat proteins. The folding of repeat proteins has been shown to follow a 1-D Ising model, wherein fixed values for intra- and inter-repeat stability combined with the number of repeats are used to define the free energy of unfolding. Nevertheless, some questions remained unanswered, and we are using consensus-designed tetratricopeptide repeats (CTPRs) to address them: How are CTPR proteins able to remain soluble and stable without capping repeats? What role does the loop between adjacent repeats play in this folding paradigm? How do the loops contribute to inter-repeat stability? Here we explore the role of loops in the thermodynamics and kinetics of repeat protein folding by inserting variable-length unstructured loops at single or multiple sites along the repeat array. In particular we seek to understand how long loops are accommodated in the current folding models and to what extent the repeat protein is able to close these loops efficiently and still fold correctly. We also want to assess the feasibility of inserting functional peptide/protein motifs in between the stable inter-repeat interfaces so that we can expand the scope for exploiting repeat proteins as building blocks in biomaterials and the controlled geometric arrangement of function within them. Our preliminary results show that CTPR proteins with multiple loop insertions are stable and correctly folded. However, we find that stability no longer increases with the number of repeats, in contrast to what is observed in normal CTPR proteins and in striking deviation from the repeat protein folding paradigm. Further analysis is required to understand this breakdown in folding cooperativity and the implications for protein design.

Exploring Folding Cooperativity of a Repeat Protein Folding by 2D-NMR Detected Pressure Perturbation

Most natural proteins fold cooperatively to form their tertiary structure. Repeat proteins offer a good model for the study of the molecular determinants of folding cooperativity because of their linear nature. In this study, we measured the pressure-induced unfolding of the leucine-rich repeat protein, pp32, at four different temperatures, using high pressure 2D NMR spectroscopy. Pressure leads to protein unfolding because internal cavities in the folded state are lost upon unfolding, thereby decreasing the molar volume of the protein-solvent system. From the pressure dependence of the individual HSQC peak intensities we obtained residue specific pressure denaturation curves. Deviations from cooperative unfolding were manifested by differences in the apparent thermodynamic parameters (ΔVu, ΔGu) extracted from fits of the data from each residue to a 2-state transition. We found the apparent unfolding cooperativity to be strongly temperature dependent. Unfolding was highly cooperative at 293°K, but deviations from two-state behavior were significant at higher and lower temperature. Then, we used fractional contact map analysis to visualize the structural basis of the unfolding heterogeneity. The fraction of contact of each residue pair was used to generate experimentally biased Structure Based Modeling (SBM) ensembles under different conditions, yielding an approximation of the folding free energy landscape. We found that at both high and low temperatures, several partially folded intermediates were populated in which the N-terminal repeats unfolded at lower pressures on average than the C-terminal repeats.

Mapping the Topography of a Protein Energy Landscape.

Protein energy landscapes are highly complex, yet the vast majority of states within them tend to be invisible to experimentalists. Here, using site-directed mutagenesis and exploiting the simplicity of tandem-repeat protein structures, we delineate a network of these states and the routes between them. We show that our target, gankyrin, a 226-residue 7-ankyrin-repeat protein, can access two alternative (un)folding pathways. We resolve intermediates as well as transition states, constituting a comprehensive series of snapshots that map early and late stages of the two pathways and show both to be polarized such that the repeat array progressively unravels from one end of the molecule or the other. Strikingly, we find that the protein folds via one pathway but unfolds via a different one. The origins of this behavior can be rationalized using the numerical results of a simple statistical mechanics model that allows us to visualize the equilibrium behavior as well as single-molecule folding/unfolding trajectories, thereby filling in the gaps that are not accessible to direct experimental observation. Our study highlights the complexity of repeat-protein folding arising from their symmetrical structures; at the same time, however, this structural simplicity enables us to dissect the complexity and thereby map the precise topography of the energy landscape in full breadth and remarkable detail. That we can recapitulate the key features of the folding mechanism by computational analysis of the native structure alone will help toward the ultimate goal of designed amino-acid sequences with made-to-measure folding mechanisms-the Holy Grail of protein folding.

StaRProtein, a web server for prediction of the stability of repeat proteins.

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins.

Resolving Cooperative Interactions in Protein Folding

Understanding the intricate relationships between protein sequence, structure, and stability remains a challenge. One major obstacle in understanding these relationships is the identification of interactions that couple elements of structure; these interactions are not readily apparent from structure. Recently, the application of nearest-neighbor models to repeat protein folding have begun to provide insights into how these systems distribute their energy within, and between units of protein structure1,2,3. Consensus ankyrin and 34-residue cTPR protein (aka c34PR) folding can be described using two terms: the intrinsic energy of individual repeats (ΔGi), and the interfacial energy between adjacent repeats (ΔGi,i+1).Here, we extend this approach in two ways using two experimental TPR-like systems. First, we dissect the whole-repeat nearest-neighbor model by representing (and resolving) the subunits of the nearest-neighbor model into each of the two helices (“A” and “B”) of each repeat. This extended Ising analysis allows us to quantify coupling energies within and between repeats, as well as local stability differences of the A and B repeat helices. Second, we explore a new, longer class of TPR-like proteins with 42 residues per repeat (42PRs, as opposed to 34 residues for the founding TPR sequence motif), to determine how variation in helix length and interface structure affects intrinsic and coupling energy.By varying the length of constructs in half-repeat increments, we find energetic heterogeneity at both the intrinsic and interfacial level. Although stabilities are more homogeneously distributed in the c34PR series (ΔGAB and ΔGBA as well as ΔGA and ΔGB are nearly isoenergetic), the distribution is more heterogeneous within 42PRs, with a greater energetic separation between intrinsic and interfacial energies. This leads to greater folding cooperativity within the 42PR series.[1] Wetzel and Pluckthun, JMB[2] Aksel, Structure[3] Kajandar et al, JACS

Intermediates in the folding equilibrium of repeat proteins from the TPR family.

In recent decades, advances in computational methods and experimental biophysical techniques have improved our understanding of protein folding. Although some of these advances have been remarkable, the structural variability of globular proteins usually encountered makes it difficult to extract general features of their folding processes. To overcome this difficulty, experimental and computational studies of the folding of repeat (or modular) proteins are of interest. Because their native structures can be described as linear arrays of the same, repeated, supersecondary structure unit, it is possible to seek a possibly independent behavior of the different modules without taking into account the intrinsic stability associated with different secondary structure motifs. In this work we have used a Monte Carlo-based simulation to study the folding equilibrium of four repeat proteins belonging to the tetratricopeptide repeat family. Our studies provide new insights into their energy profiles, enabling investigation about the existence of intermediate states and their relative stabilities. We have also performed structural analyses to describe the structure of these intermediates, going through the vast number of conformations obtained from the simulations. In this way, we have tried to identify the regions of each protein in which the modular structure yields a different behavior and, more specifically, regions of the proteins that can stay folded when the rest of the chain has been thermally denatured.

The Contribution of Entropy, Enthalpy, and Hydrophobic Desolvation to Cooperativity in Repeat-Protein Folding

Cooperativity is a defining feature of protein folding, but its thermodynamic and structural origins are not completely understood. By constructing consensus ankyrin repeat protein arrays that have nearly identical sequences, we quantify cooperativity by resolving stability into intrinsic and interfacial components. Heteronuclear NMR and CD spectroscopy show that these constructs adopt ankyrin repeat structures. Applying a one-dimensional Ising model to a series of constructs chosen to maximize information content in unfolding transitions, we quantify stabilities of the terminal capping repeats, and resolve the effects of denaturant into intrinsic and interfacial components. Reversible thermal denaturation resolves interfacial and intrinsic free energies into enthalpic, entropic, and heat capacity terms. Intrinsic folding is entropically disfavored, whereas interfacial interaction is entropically favored and attends a decrease in heat capacity. These results suggest that helix formation and backbone ordering occurs upon intrinsic folding, whereas hydrophobic desolvation occurs upon interfacial interaction, contributing to cooperativity.

Determinants of Cooperativity in Repeat Protein Folding

Repeat proteins have been popular model systems in protein folding research due to their modular architecture. Although each repeat has the same fold, because of sequence variation, the folding energy is not distributed evenly among the repeats. Therefore, it is challenging to quantify the repeat-repeat interactions and the intrinsic folding energies of every repeat. To overcome this difficulty, we designed a consensus ankyrin repeat(CANK), which is brought together to make arrays of identical repeats. By solution NMR, we solved the structure of our three repeat CANK, NRC, and verify that NRC adopts the canonical Ankyrin repeat protein fold. Our SAXS measurements show that CANKs in different length arrays also adopt the same fold. By fitting unfolding transitions of a limited number of constructs to Ising model, we found that the individual repeats have low intrinsic stability, but the interfacial interactions are highly stabilizing. We also extended our Ising formalism to include the temperature dependence of unfolding free energy and we quantified the enthalpic and entropic contribution to intrinsic and interfacial stabilities, as well as, the uneven distribution of heat capacity of unfolding into the intrinsic and interfacial components. Our results suggest that intrinsic folding resembles secondary structure formation(loss of conformational entropy) and interfacial folding involves hydrophobic desolvation(entropy increase and negative heat capacity change upon folding). To separate out the contribution of long range electrostatics to intrinsic and interfacial stability, we measured the salt dependence of CANK stabilities. Our results show that NaCl stabilizes the interfaces and has marginal destabilizing effect on intrinsic stability. Currently we are trying to identify the charge-charge interactions responsible for the high salt dependence of interfacial stability by NMR techniques and single point substitutions.

Folding Studies of Beta-Strand-Containing Repeat Proteins Through Naturally-Occurring and Consensus-Designed Sequences

Repeat proteins, devoid of sequence-distant contacts observed in globular proteins, are ideal candidates for the dissection of local stability, nearest-neighbor contact parameters, cooperativity, and determination of folding energy landscapes. Recent studies on HEAT, TPR, Ankyrin, and Armadillo have contributed to the understanding of the folding of helical repeat proteins. Moreover, constructs composed of simplified “consensus” sequence representations of helical repeat proteins have been found to be well-behaved and highly stable, and facilitate energy dissection using simple nearest-neighbor models. In contrast, little progress has been made towards understanding the folding of β-sheet containing repeat. To determine the folding stability and cooperativity of these proteins, and to better understand the sequence determinants of structure and stability within these ubiquitous families, we have initiated studies on a series of LRR proteins of both naturally occurring and consensus-designed sequences. We find the LRR proteins PP32 and LC1 to be well-behaved, and to fold in a highly cooperative transition that is consistent with a two-state mechanism. NMR H/2H exchange shows the repeating β-strands on the concave surface of both proteins to be more protected than the rest of the molecules, and can be regarded as an exchange-resistant core, whereas the terminal caps and convex structural elements are more labile. However, truncations and sequence substitution demonstrate that the caps significantly influence stability and kinetics. To further simplify our analysis of β-sheet containing repeat protein folding, we designed consensus LRR sequences. On their own, these constructs are unfolded and/or aggregated. By fusing these consensus sequences with naturally occurring LRR protein YopM, we have obtained solublized, folded arrays that exhibit increased stability and drastically decreased unfolding and refolding rates with repeat number. Further studies are needed to dissect the complex folding pathways taken by these constructs.

Full Reconstruction of a Vectorial Protein Folding Pathway by Afm and Smd: Insights Into the Co-Translational Folding of the Nascent-Polypeptide-Chain

During co-translational folding, the nascent-polypeptide-chain (NPC) is extruded sequentially from the ribosome exit tunnel and under severe conformational constraints dictated by its 1D geometry. Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of 1D constraints that are similar to those imposed on the NPC. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regained structures without any nucleation step, by using the intact N terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.

Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and is [corrected] under severe conformational constraints [corrected] dictated by the one-dimensional geometry of the tunnel. [corrected] How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.