Cooperative folding kinetics of BBL protein and peripheral subunit-binding domain homologues

Recent experiments claiming that Naf-BBL protein follows a global downhill folding raised an important controversy as to the folding mechanism of fast-folding proteins. Under the global downhill folding scenario, not only do proteins undergo a gradual folding, but folding events along the continuous folding pathway also could be mapped out from the equilibrium denaturation experiment. Based on the exact calculation using a free energy landscape, relaxation eigenmodes from a master equation, and Monte Carlo simulation of an extended Muñoz–Eaton model that incorporates multiscale-heterogeneous pairwise interactions between amino acids, here we show that the very nature of a two-state cooperative transition such as a bimodal distribution from an exact free energy landscape and biphasic relaxation kinetics manifest in the thermodynamics and folding–unfolding kinetics of BBL and peripheral subunit-binding domain homologues. Our results provide an unequivocal resolution to the fundamental controversy related to the global downhill folding scheme, whose applicability to other proteins should be critically reexamined.     

Dynamics of one-state downhill protein folding

The small helical protein BBL has been shown to fold and unfold in the absence of a free energy barrier according to a battery of quantitative criteria in equilibrium experiments, including probe-dependent equilibrium unfolding, complex coupling between denaturing agents, characteristic DSC thermogram, gradual melting of secondary structure, and heterogeneous atom-by-atom unfolding behaviors spanning the entire unfolding process. Here, we present the results of nanosecond T-jump experiments probing backbone structure by IR and end-to-end distance by FRET. The folding dynamics observed with these two probes are both exponential with common relaxation times but have large differences in amplitude following their probe-dependent equilibrium unfolding. The quantitative analysis of amplitude and relaxation time data for both probes shows that BBL folding dynamics are fully consistent with the one-state folding scenario and incompatible with alternative models involving one or several barrier crossing events. At 333 K, the relaxation time for BBL is 1.3 μs, in agreement with previous folding speed limit estimates. However, late folding events at room temperature are an order of magnitude slower (20 μs), indicating a relatively rough underlying energy landscape. Our results in BBL expose the dynamic features of one-state folding and chart the intrinsic time-scales for conformational motions along the folding process. Interestingly, the simple self-averaging folding dynamics of BBL are the exact dynamic properties required in molecular rheostats, thus supporting a biological role for one-state folding.     

Structural insights into an equilibrium folding intermediate of an archaeal ankyrin repeat protein

Repeat proteins are widespread in nature, with many of them functioning as binding molecules in protein-protein recognition. Their simple structural architecture is used in biotechnology for generating proteins with high affinities to target proteins. Recent folding studies of ankyrin repeat (AR) proteins revealed a new mechanism of protein folding. The formation of an intermediate state is rate limiting in the folding reaction, suggesting a scaffold function of this transient state for intrinsically less stable ARs. To investigate a possible common mechanism of AR folding, we studied the structure and folding of a new thermophilic AR protein (tANK) identified in the archaeon Thermoplasma volcanium. The x-ray structure of the evolutionary much older tANK revealed high homology to the human CDK inhibitor p19(INK4d), whose sequence was used for homology search. As for p19(INK4d), equilibrium and kinetic folding analyses classify tANK to the family of sequential three-state folding proteins, with an unusual fast equilibrium between native and intermediate state. Under equilibrium conditions, the intermediate can be populated to >90%, allowing characterization on a residue-by-residue level using NMR spectroscopy. These data clearly show that the three C-terminal ARs are natively folded in the intermediate state, whereas native cross-peaks for the rest of the molecule are missing. Therefore, the formation of a stable folding unit consisting of three ARs is the necessary rate-limiting step before AR 1 and 2 can assemble to form the native state.     

Structure and folding of a designed knotted protein

A very small number of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophysical characterization and X-ray crystal structure determination show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approximately 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic experiments suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy--connecting two monomers of an intertwined homodimer into a single protein chain--supports a model for evolution of knotted structures via gene duplication.     

The nature of protein folding pathways

How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the “new view” model for protein folding. Emergent macroscopic foldon–foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the “how” and the “why” questions. The protein folding pathway depends on the same foldon units and foldon–foldon interactions that construct the native structure.     

Role of structural determinants in folding of the sandwich-like protein Pseudomonas aeruginosa azurin

An invariant substructure that forms two interlocked pairs of neighboring beta-strands occurs in essentially all known sandwich-like proteins. Eight conserved positions in these strands were recently shown to act as structural determinants. To test whether the residues at these invariant positions are conserved for mechanistic (i.e., part of folding nucleus) or energetic (i.e., governing native-state stability) reasons, we characterized the folding behavior of eight point-mutated variants of the sandwich-like protein Pseudomonas aeruginosa apo-azurin. We find a simple relationship among the conserved positions: half of the residues form native-like interactions in the folding transition state, whereas the others do not participate in the folding nucleus but govern high native-state stability. Thus, evolutionary preservation of these specific positions gives both mechanistic and energetic advantages to members of the sandwich-like protein family.     

Topological constraints and modular structure in the folding and functional motions of GlpG,an intramembrane protease

We investigate the folding of GlpG, an intramembrane protease, using perfectly funneled structure-based models that implicitly account for the absence or presence of the membrane. These two models are used to describe, respectively, folding in detergent micelles and folding within a bilayer, which effectively constrains GlpG''s topology in unfolded and partially folded states. Structural free-energy landscape analysis shows that although the presence of multiple folding pathways is an intrinsic property of GlpG’s modular functional architecture, the large entropic cost of organizing helical bundles in the absence of the constraining bilayer leads to pathways that backtrack (i.e., local unfolding of previously folded substructures is required when moving from the unfolded to the folded state along the minimum free-energy pathway). This backtracking explains the experimental observation of thermodynamically destabilizing mutations that accelerate GlpG’s folding in detergent micelles. In contrast, backtracking is absent from the model when folding is constrained within a bilayer, the environment in which GlpG has evolved to fold. We also characterize a near-native state with a highly mobile transmembrane helix 5 (TM5) that is significantly populated under folding conditions when GlpG is embedded in a bilayer. Unbinding of TM5 from the rest of the structure exposes GlpG’s active site, consistent with studies of the catalytic mechanism of GlpG that suggest that TM5 serves as a substrate gate to the active site.GlpG is a rhomboid protease that sits and functions in the cell membrane. GlpG’s homologs are found across all kingdoms of life. GlpG has been the subject of several biophysical experimental studies aimed toward understanding membrane protein folding and the relationships among protein structure, dynamics, and function (1–5). An extensive experimental φ-value analysis found φ-values significantly different from zero, indicative of structural changes during the rate-limiting step of folding, in transmembrane helices 1 through 5 (TM1-5) and the intervening loops (4). Most of the nonzero φ-values, particularly in TM3-5 and in the large loop L1, were negative, meaning that although the corresponding mutation destabilizes the native state, the mutation nonetheless accelerates folding. The preponderance of negative φ-values was puzzling and unprecedented, and at the time, these effects were tentatively ascribed to nonnative interactions in the transition state ensemble. In this work, we show that, in fact, simple models with perfectly funneled energy landscapes that lack nonnative interactions are able to explain the origin of these negative φ-values and how the values arise when folding in detergent micelles rather than bilayers.α-Helical membrane protein folding is thought to occur in two stages in vivo (6). The first stage, setting up the proper topology of transmembrane helices, is handled by the translocon (7, 8). In the present context, topology refers to specifying the directions in which a membrane protein’s constituent transmembrane helices traverse the bilayer. The second stage, converting from properly inserted but dissociated helices into a functional folded structure, occurs spontaneously and is, in some ways, analogous to soluble protein folding. However, we know, ranging from the hydrophobic effect (9, 10) to water-mediated (11) and screened electrostatic interactions (12), the solvent plays a role in determining what types of noncovalent interactions are stabilizing and destabilizing. Whereas soluble proteins fold in polar and isotropic aqueous solutions, membrane proteins fold in largely apolar and anisotropic environments. These environmental differences complicate applying directly methods developed for studying soluble protein folding to the study of membrane protein folding. Nonetheless, experimentalists have been able to apply a variety of methods to study the kinetics and thermodynamics of membrane protein folding through the use of detergent micelles as a membrane-mimicking environment. Experiments that probe the folding mechanisms of membrane proteins have used micelles composed of a mixture of anionic and nonionic detergents (4, 13, 14), which not only keep membrane proteins soluble but also, through use of mixed micelles, allow the equilibrium between folded and unfolded states to be tuned. Micelles predominantly composed of nonionic detergents, such as n-dodecyl-β-d-maltopyranoside (DDM), preferentially stabilize a folded state that has been shown to be functional and is therefore likely to be structurally similar to the folded state in vivo. Micelles predominantly composed of anionic detergents, on the other hand, preferentially stabilize an unfolded state that contains significant amounts of secondary structure. This ability to tune the equilibrium means that stopped-flow kinetic experiments can be combined with protein-engineering techniques to determine folding mechanisms at the single-residue level (4, 13, 15), in analogy to what has been done for soluble proteins (16–18). Because carrying out these types of experiments in bilayers is still difficult, it is presently unknown how folding mechanisms determined in micelles compare with those in membranes. Confining proteins to a 2D membrane is expected to constrain unfolded and partially folded ensembles to having structures with helices that are largely properly aligned and embedded in the membrane; such topological restrictions would be relaxed in a micellar environment.Theoretical (19, 20) and experimental (3, 4) work suggests that at least some membrane proteins can reversibly fold and unfold without the aid of the translocon or chaperones in vitro. It is therefore likely that membrane protein folding landscapes are funneled, much like globular protein landscapes (21, 22). Structure-based models with perfectly funneled energy landscapes have proven useful for investigating the folding and binding of proteins (23, 24). In this study, we use a structure-based model to investigate folding of a membrane protein in two different situations: in the absence and presence of an implicit membrane energy term that biases conformations to have the correct topology with respect to the membrane. Simulations with the implicit membrane term are thus taken to model folding in a bilayer, whereas simulations without the implicit membrane energy are taken to model folding in detergent micelles. Although this way of modeling micelles and bilayers is an oversimplification, it captures the significantly increased topological freedom of membrane proteins in micellar environments compared with lipid bilayer. Fig. 1 shows schematic representations of the corresponding denatured states of membrane proteins in bilayers and micelles.Open in a separate windowFig. 1.Schematic diagrams of the unfolded state of α-helical membrane proteins in bilayers (Left) and detergent micelles (Right). The transmembrane helices (cylinders) are connected by loops. Transmembrane helices are either embedded in a membrane (rectangular prism) or are surrounded by detergent micelles (transparent gray spheres). In this work, we use an implicit membrane model to simulate folding within a bilayer and assume that folding in detergent micelles corresponds to folding without constraints on the alignment of helices. In both cases, we assume that the unfolded state has near-native levels of secondary structure, as has been observed in experiments on the SDS-denatured state of membrane proteins.The same energy landscape that dictates folding routes also encodes functional motions. It has been suggested that the modularity in the structure of GlpG supports functional motions (1, 25). The N-terminal domain, which contains transmembrane helices 1 and 2 (TM1-2) as well as the intervening L1 loop, functions as a structural scaffold (25), whereas the C-terminal domain with its four transmembrane helices (TM3-6) includes the catalytic site (25). The C-terminal domain is apparently more flexible than the N-terminal domain; both the loop L5 (5) and the transmembrane helix TM5 (25) have been crystallized in multiple conformations. Because of this flexibility, it has been suggested that either L5 alone (5) or L5 and TM5 (25) may serve as a substrate gate for access to the catalytic site. Using free-energy landscape analysis and perturbation methods along with structural analysis, we show that there is a near-native state significantly populated under folding conditions and elucidate the state’s connections to GlpG’s folding mechanism and function.     

A thermodynamic definition of protein domains

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.     

Stable folding core in the folding transition state of an alpha-helical integral membrane protein

Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G.     

Simulation of Top7-CFr: a transient helix extension guides folding

Protein structures often feature beta-sheets in which adjacent beta-strands have large sequence separation. How the folding process orchestrates the formation and correct arrangement of these strands is not comprehensively understood. Particularly challenging are proteins in which beta-strands at the N and C termini are neighbors in a beta-sheet. The N-terminal beta-strand is synthesized early on, but it can not bind to the C terminus before the chain is fully synthesized. During this time, there is a danger that the beta-strand at the N terminus interacts with nearby molecules, leading to potentially harmful aggregates of incompletely folded proteins. Simulations of the C-terminal fragment of Top7 show that this risk of misfolding and aggregation can be avoided by a "caching" mechanism that relies on the "chameleon" behavior of certain segments.     

Modeling transient collapsed states of an unfolded protein to provide insights into early folding events

The primary driving force for protein folding is the sequestration of hydrophobic side chains from solvent water, but the means whereby the amino acid sequence directs the folding process to form the correct final folded state is not well understood. Measurements of NMR line broadening in spin-labeled samples of unfolded apomyoglobin at pH 2.3 have been used to derive a quantitative model for transient hydrophobic interactions between various sites in the polypeptide chain, as would occur during the initiation of protein folding. Local clusters of residues with high values for the parameter "average area buried upon folding" (AABUF) form foci not only for local contacts but for long-range interactions, the relative frequencies of which can be understood in terms of differences in the extent of reduction in chain configurational entropy that occurs upon formation of nonlocal contacts. These results complement the striking correlation previously observed between the kinetic folding process of apomyoglobin and the AABUF of its amino acid sequence [Nishimura C, Lietzow MA, Dyson HJ, Wright PE (2005) J Mol Biol 351:383-392]. For the acid-unfolded states of apomyoglobin, our approach identifies multiple distinct hydrophobic clusters of differing thermodynamic stability. The most structured of these clusters, although sparsely populated, have both native-like and nonnative character; the specificity of the transient long-range contacts observed in these states suggests that they play a key role in initiating chain collapse and folding.     

Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding

Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier. This protein is very stable and topologically simple, needing merely to "zipper up," while having an extrapolated folding rate of k(f) = 2 x 10(5) s(-1). These properties make it likely to attain the elusive "downhill folding" limit, at which a series of intermediates can be characterized. To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods. The stability and its denaturant dependence for the hydrogen bonds in the central part of protein equal the values calculated for whole-molecule unfolding. Like-wise, their closing and opening rates indicate that these hydrogen bonds are broken and reformed in a single cooperative event representing the folding transition from the fully unfolded state to the native state. Additionally, closing rates for these hydrogen bonds agree with the extrapolated barrier-limited folding rate observed near the melting transition. Therefore, even in the absence of denaturant, where DeltaG(eq) approximately -6 kcal.mol(-1) (1 cal = 4.18 J) and tau(f) approximately 6 mus, folding remains cooperative and barrier-limited. Given that this prime candidate for downhill folding fails to do so, we propose that protein folding will remain barrier-limited for proteins that fold cooperatively.     

Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation.     

Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.

Transmembrane α-helices are the basic structural principle of most membrane proteins. As a result, the topology of multispanning membrane proteins is defined by a succession of helices of alternating orientation, separated by loops exposed to the cytoplasm and to the exoplasmic space [i.e., the lumen of the endoplasmic reticulum (ER) in eukaryotes or the exterior of the plasma membrane in prokaryotes (1)]. Upon integration, the transmembrane segments assemble to a helix bundle, while the loops fold in either the cytosol or the exoplasmic space, where chaperones may assist to prevent misfolding and aggregation.The integration of transmembrane domains and translocation of exoplasmic loops are mediated by the conserved Sec61/SecY translocon composed of Sec61-αβγ in the ER of eukaryotes and SecYEG in prokaryotes (2). The main subunit Sec61-α/SecY is a 10-transmembrane helix bundle that can open a pore across the membrane for the translocation of hydrophilic polypeptide chains and a lateral gate for the exit of hydrophobic segments into the lipid environment. In its idle state, the translocon is closed and stabilized by a short helix that forms an exoplasmic plug and by a central constriction.Proteins are targeted to the membrane by a hydrophobic signal sequence that, as it emerges from the ribosome, binds to signal recognition particle (SRP). The signal is either an amino-terminal cleavable signal peptide or the first transmembrane domain of the protein acting as an uncleaved signal anchor. SRP targets the translating ribosome to the SRP receptor and to the translocon. The ribosome binds to cytoplasmic loops of the C-terminal, half of the translocon, leaving a gap open toward the cytosol (3). As the protein is synthesized, its transmembrane segments are inserted successively into the lipid bilayer.Three distinct membrane integration processes can be distinguished (schematically shown in Fig. 1A). First, the signal (anchor) activates the translocon by intercalating between the gate helices and exiting toward the lipid phase (2). In the process, the hydrophilic flanking sequence is inserted into the pore for transfer into the lumen, and the plug is pushed away. This has been elucidated by a number of structures of translocons engaged with signal sequences (3–5). Which end of a signal anchor will be translocated is mainly determined by the flanking charges [the “positive–inside rule” states that the more positive end will be cytosolic (6–8)] and the folding state of the N-terminal domain hindering translocation (9). As the nascent chain is moving through the translocon pore, a hydrophobic transmembrane segment stops further transfer by leaving via the lateral gate into the lipid membrane as a stop-transfer sequence. The following polypeptide is then synthesized through the gap between ribosome and translocon into the cytosol until a further transmembrane segment again engages with the translocon, exits into the membrane, and inserts the downstream sequence into the pore. This reintegration process is similar to the initial signal integration. Furthermore, alternating stop-transfer and reintegration events may account for any number of transmembrane domains.Open in a separate windowFig. 1.Reintegration efficiency is affected by the cytoplasmic upstream sequence. (A) Schematic representation of the three distinct integration processes in the biogenesis of membrane proteins: signal (anchor) integration (red transmembrane segment) (1), stop-transfer integration (blue) (2), and reintegration (green) (3). Transmembrane domains do not necessarily fully enter the translocon, before exiting into the membrane, but may associate with lipids early on and glide along the gate, as proposed by Cymer et al. (43). (B) The construct RI-DP128 was derived from wt DPAPB via ST-DP128 (DPAPB-H in ref. 12), as shown schematically with the transmembrane domains in red, blue, and green as in A. Rings indicate potential N-glycosylation sites. A C-terminal triple–HA-tag is shown in gray. To analyze the hydrophobicity threshold of membrane integration, the H segment sequence GGPGAAAAAAAAAAAAAAAAAAAGPGG, with 0 to 19 alanines replaced by leucines, were inserted as a potential reintegration sequence (green) in RI-DP. Below, the DPAPB sequences of the cytosolic loop between the stop-transfer and the reintegration domains of RI-DP128 and the deletion constructs RI-DP100–22 are listed, with a dash indicating the deletion site. At least 20 residues preceding the reintegration H segment were kept constant in all constructs. (C) Schematic representation of the two topologies when the H segments do or do not initiate reintegration and C-terminal translocation. cyt, cytosol; exo, exoplasm = ER lumen. Glycosylations are indicated by Y. (D) The various constructs, each with different reintegration H segments composed of 19 alanines, with 0 to 19 of them replaced by leucine residues (L#), were expressed in yeast cells, labeled with [³⁵S]methionine for 5 min, immunoprecipitated, and analyzed by SDS–gel electrophoresis and autoradiography. Based on the glycosylation pattern, integration (I) or nonintegration (N) of the H segment, as well as uninserted unglycosylated products (U) were distinguished. To identify the position of the unglycosylated polypeptide, a sample was analyzed after deglycosylation by endoglycosidase H (H) on the right. (E) The fraction of products with reintegrated H segments as a percentage of the total membrane-integrated proteins was plotted versus the number of leucine residues in the H segment (mean, SD, and the individual values of at least three independent experiments). The curves are labeled with the length of the constructs’ cytosolic loops between stop transfer and H segment. (F) The cytoplasmic sequence between stop transfer and H segment is shown for RI-CP145 (residues 21 to 160 of pre-CPY, in gray) and RI-CP22 (residues 144 to 160). (G) The CPY constructs were expressed and analyzed, as in D. (H) Reintegration of the H segment was quantified, as in E (mean, SD, and the individual values of at least three independent experiments).The main characteristic of transmembrane segments is their overall hydrophobicity on a length of ∼20 residues that are required for an α-helix to span the apolar core of a bilayer. This was confirmed by systematic quantitative analysis of a large number of so-called H segments, mildly hydrophobic sequences of 19 residues, for their efficiency as stop-transfer sequences (10). From these experiments, a “biological hydrophobicity scale” for all 20 amino acids was derived. The dependence of integration efficiency on hydrophobicity is consistent with a purely thermodynamic equilibration process between the pore interior and the lipid environment for each peptide segment entering the translocon. This concept allowed for the estimation of apparent free energy contributions ∆G^aa_app of each amino acid to membrane integration. The contribution of particular amino acids also depended on their position in a manner reflecting the structure of the membrane (11): Polarizable residues, such as tryptophan or tyrosine, energetically favor the polarity interface between apolar fatty acyl and polar lipid head group regions, while polar and charged residues are energetically most unfavorable in the center of the bilayer, thus reducing integration efficiency.The equilibration model is further supported by mutagenesis of the translocon. The six residues forming the central constriction of the translocation pore are predominantly hydrophobic. Their mutation to more hydrophilic amino acids, to increase polarity and hydration inside the pore, reduced the hydrophobicity threshold for membrane integration considerably (12, 13), as expected for a partitioning process.From these findings emerges the concept that each sequence of ∼20 residues, based on its amino acids and their position within the segment, autonomously defines its propensity to integrate into the membrane. The data on the energetics of membrane integration was mostly collected for stop-transfer integration at the mammalian ER. Yet similar hydrophobicity dependence and hydrophobicity scales for integration were determined in other systems, such as for stop transfer in yeast (12, 14) or bacteria (15, 16), for reintegration in yeast and mammalian ER (17) and even for TIM23-mediated integration into the inner mitochondrial membrane (18). The biological hydrophobicity scale thus is considered to be universal and generally applicable to transmembrane segments, irrespective of their orientation, the organism, or the compartment. Accordingly, it is also used for topology prediction from primary sequences (19).However, the different systems that were analyzed also showed significant quantitative shifts in the hydrophobicity threshold for integration. A simple hydrophobicity series, as initially introduced by Hessa et al. (10), is produced with H segments consisting of 19 alanines, of which 0 to 19 are replaced by leucine. The threshold for 50% stop-transfer integration was determined to be 3.1 leucines using mammalian in vitro translation with dog pancreas microsomes and 3.8 leucines in baby hamster kidney cells (10). Further studies (summarized in ref. 1) measured thresholds between one and seven leucines [for example, 4.4 and 3.5 leucines in yeast (12, 14), 1.1 and 2.0 in Escherichia coli (15, 16), and 7.3 in HeLa cells (20)]. Using the ∆G_app values from Hessa et al. (11), this covers a range of ∼3 kcal/mol. Different lipid compositions and translocon sequences, but also the reporter constructs, may contribute. As to the latter, it has indeed been observed that the sequence immediately following an H segment can influence integration efficiency, most likely by conformationally hindering or facilitating stop-transfer release into the bilayer (21). This already challenged to some extent the autonomy by which each sequence determines its integration propensity.There is only one study that analyzed the hydrophobicity threshold of reintegration sequences (17). Surprisingly, it was found to be considerably lower than for stop-transfer integration with 0.9 versus 3.1 leucines in a mammalian in vitro translation/membrane insertion system and with 2.2 versus 4.4 leucines in yeast cells. In the present study, we set out to test the reintegration efficiency in Saccharomyces cerevisiae using the same H segments in a different reporter construct. Our results revealed a surprising dependence of reintegration on the length and/or characteristics of the sequence preceding the reintegration domain. A similar dependence was also found for stop-transfer integration. The data indicate that the folding properties of the cytoplasmic and luminal loop sequences in membrane proteins determine the integration efficiency of subsequent transmembrane domains.     

Consequences of localized frustration for the folding mechanism of the IM7 protein

In the laboratory, IM7 has been found to have an unusual folding mechanism in which an "on-pathway" intermediate with nonnative interactions is formed. We show that this intermediate is a consequence of an unusual cluster of highly frustrated interactions in the native structure. This cluster is involved in the binding of IM7 to its target, Colicin E7. Redesign of residues in this cluster to eliminate frustration is predicted by simulations to lead to faster folding without the population of an intermediate ensemble.     

A designed protein as experimental model of primordial folding

How do proteins accomplish folding during early evolution? Theoretically the mechanism involves the selective stabilization of the native structure against all other competing compact conformations in a process that involves cumulative changes in the amino acid sequence along geological timescales. Thus, an evolved protein folds into a single structure at physiological temperature, but the conformational competition remains latent. For natural proteins such competition should emerge only near cryogenic temperatures, which places it beyond experimental testing. Here, we introduce a designed monomeric miniprotein (FSD-1ss) that within biological temperatures (330–280 K) switches between simple fast folding and highly complex conformational dynamics in a structurally degenerate compact ensemble. Our findings demonstrate the physical basis for protein folding evolution in a designed protein, which exhibits poorly evolved or primordial folding. Furthermore, these results open the door to the experimental exploration of primitive folding and the switching between alternative protein structures that takes place in evolutionary branching points and prion diseases, as well as the benchmarking of de novo design methods.     

The transition state for folding of an outer membrane protein

Inspired by the seminal work of Anfinsen, investigations of the folding of small water-soluble proteins have culminated in detailed insights into how these molecules attain and stabilize their native folds. In contrast, despite their overwhelming importance in biology, progress in understanding the folding and stability of membrane proteins remains relatively limited. Here we use mutational analysis to describe the transition state involved in the reversible folding of the β-barrel membrane protein PhoPQ-activated gene P (PagP) from a highly disordered state in 10 M urea to a native protein embedded in a lipid bilayer. Analysis of the equilibrium stability and unfolding kinetics of 19 variants that span all eight β-strands of this 163-residue protein revealed that the transition-state structure is a highly polarized, partly formed β-barrel. The results provide unique and detailed insights into the transition-state structure for β-barrel membrane protein folding into a lipid bilayer and are consistent with a model for outer membrane protein folding via a tilted insertion mechanism.     

Full distance-resolved folding energy landscape of one single protein molecule

Kinetic bulk and single molecule folding experiments characterize barrier properties but the shape of folding landscapes between barrier top and native state is difficult to access. Here, we directly extract the full free energy landscape of a single molecule of the GCN4 leucine zipper using dual beam optical tweezers. To this end, we use deconvolution force spectroscopy to follow an individual molecule’s trajectory with high temporal and spatial resolution. We find a heterogeneous energy landscape of the GCN4 leucine zipper domain. The energy profile is divided into two stable C-terminal heptad repeats and two less stable repeats at the N-terminus. Energies and transition barrier positions were confirmed by single molecule kinetic analysis. We anticipate that deconvolution sampling is a powerful tool for the model-free investigation of protein energy landscapes.