Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes

Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of an alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

Design and Structure of an Equilibrium Protein Folding Intermediate: A Hint into Dynamical Regions of Proteins

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity.     

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

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

A hydrogen bond regulates slow motions in ubiquitin by modulating a β-turn flip

Proteins exist as conformational ensembles composed of multiple interchanging substates separated by kinetic barriers. Interconverting conformations are often difficult to probe, owing to their sparse population and transient nature. Here, we report the identification and characterization of a subset of conformations in ubiquitin that participate in microsecond-to-millisecond motions in the amides of Ile23, Asn25, and Thr55. A novel side chain to the backbone hydrogen bond that regulates these motions has also been identified. Combining our NMR studies with the available X-ray data, we have unearthed the physical process underlying slow motions—the interconversion of a type I into a type II β-turn flip at residues Glu51 through Arg54. Interestingly, the dominant conformer of wild-type ubiquitin observed in solution near neutral pH is only represented by about 22% of the crystal structures. The conformers generated as a result of the dynamics of the hydrogen bond appear to be correlated to ligand recognition by ubiquitin.     

Conservation of transition state structure in fast folding peripheral subunit-binding domains

Φ-Value analysis was used to characterise the structure of the transition state (TS) for folding of POB L146A Y166W, a peripheral subunit-binding domain that folds in microseconds. Helix 2 was structured in the TS with consolidating interactions from the structured loop that connects the two α-helices. This distribution of Φ-values was very similar to that determined for E3BD F166W, a homologue with high sequence and structural similarity. The extrapolated folding rate constants in water at 298 K were 210,000 s^− 1 for POB and 27,500 s^− 1 for E3BD. A contribution to the faster folding of POB came from its having significantly greater helical propensity in helix 2, the folding nucleus. The folding rate also appeared to be influenced by differences in the sequence and structural properties of the loop connecting the two helices. Unimodal downhill folding has been proposed as a conserved, biologically important property of peripheral subunit-binding domains. POB folds five times faster and E3BD folds slower than a proposed limit of 40,000 s^− 1 for barrier-limited folding. However, experimental evidence strongly suggests that both POB L146A Y166W and E3BD F166W fold in a barrier-limited process through a very similar TS ensemble.     

Phi-analysis of the folding of the B domain of protein A using multiple optical probes

We examined the co-operativity of ultra-fast folding of a protein and whether the Phi-value analysis of its transition state depended on the location of the optical probe. We incorporated in turn a tryptophan residue into each of the three helices of the B domain of Protein A. Each Trp mutant of the three-helix bundle protein was used as a pseudo-wild-type parent for Phi-analysis in which the intrinsic Trp fluorescence probed the formation of each helix during the transition state. Apart from local effects in the immediate vicinity of the probe, the three separate sets of Phi-values were in excellent agreement, demonstrating the overall co-operativity of folding and the robustness of the Phi-analysis. The transition state of folding of Protein A contains the second helix being well formed with many stabilizing tertiary hydrophobic interactions. In contrast, the first and the third helices are more poorly structured in the transition state. The mechanism of folding thus involves the concurrent formation of secondary and tertiary interactions, and is towards the nucleation-condensation extreme in the nucleation-condensation-framework continuum of mechanism, with helix 2 being the nucleus. We provide an error analysis of Phi-values derived purely from the kinetics of two-state chevron plots.     

Structure, Dynamics and Folding of an Immunoglobulin Domain of the Gelation Factor (ABP-120) from Dictyostelium discoideum

We have carried out a detailed structural and dynamical characterisation of the isolated fifth repeat of the gelation factor (ABP-120) from Dictyostelium discoideum (ddFLN5) by NMR spectroscopy to provide a basis for studies of co-translational folding on the ribosome of this immunoglobulin-like domain. The isolated ddFLN5 can fold autonomously in solution into a structure that resembles very closely the crystal structure of the domain in a construct in which the adjacent sixth repeat (ddFLN6) is covalently linked to its C-terminus in tandem but deviates locally from a second crystal structure in which ddFLN5 is flanked by ddFLN4 and ddFLN6 at both N- and C-termini. Conformational fluctuations were observed via ¹⁵N relaxation methods and are primarily localised in the interstrand loops that encompass the C-terminal hemisphere. These fluctuations are distinct in location from the region where line broadening is observed in ddFLN5 when attached to the ribosome as part of a nascent chain. This observation supports the conclusion that the broadening is associated with interactions with the ribosome surface [Hsu, S. T. D., Fucini, P., Cabrita, L. D., Launay, H., Dobson, C. M. & Christodoulou, J. (2007). Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy. Proc. Natl. Acad. Sci. USA, 104, 16516-16521]. The unfolding of ddFLN5 induced by high concentrations of urea shows a low population of a folding intermediate, as inferred from an intensity-based analysis, a finding that differs from that of ddFLN5 as a ribosome-bound nascent chain. These results suggest that interesting differences in detail may exist between the structure of the domain in isolation and when linked to the ribosome and between protein folding in vitro and the folding of a nascent chain as it emerges from the ribosome.     

Downhill versus Barrier-Limited Folding of BBL 1: Energetic and Structural Perturbation Effects upon Protonation of a Histidine of Unusually Low pKa

A dispersion of melting temperatures at pH 5.3 for individual residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral subunit domain family fold cooperatively at pH 7. To search for possible causes of anomalies in BBL's denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pK_a of His142 was close to that of free histidine at 6.47 ± 0.04, while that of the more buried His166 was highly perturbed at 5.39 ± 0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by ∼ 1.5 kcal/mol. Changes in C^α secondary shifts at pH 5.3 showed a decrease in helicity of the C-terminus of helix 2, where His166 is located, which was accompanied by a measured decrease of 1.1 ± 0.2 kcal/mol in stability from pH 7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only ∼ 1% of the structurally perturbed state will be present at the biologically relevant pH 7.6. Experiments at pH 5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pK_a induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH 5.3.     

Solution structure of the inner DysF domain of myoferlin and implications for limb girdle muscular dystrophy type 2b

Mutations in the protein dysferlin, a member of the ferlin family, lead to limb girdle muscular dystrophy type 2B and Myoshi myopathy. The ferlins are large proteins characterised by multiple C2 domains and a single C-terminal membrane-spanning helix. However, there is sequence conservation in some of the ferlin family in regions outside the C2 domains. In one annotation of the domain structure of these proteins, an unusual internal duplication event has been noted where a putative domain is inserted in between the N- and C-terminal parts of a homologous domain. This domain is known as the DysF domain. Here, we present the solution structure of the inner DysF domain of the dysferlin paralogue myoferlin, which has a unique fold held together by stacking of arginine and tryptophans, mutations that lead to clinical disease in dysferlin.     

Structure and dynamics of pin1 during catalysis by NMR

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the microsecond-to-millisecond timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidylprolyl isomerase (Pin1) catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer's disease. (15)N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of a substrate, suggesting that this area is already "primed" for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intramolecular nuclear Overhauser enhancements. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed the distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.     

Solution Structure of Human ζ-COP: Direct Evidences for Structural Similarity between COP I and Clathrin-Adaptor Coats

COP-I-coated vesicles are protein and lipid carriers that mediate intra-Golgi transport and transport from the cis-Golgi complex to the endoplasmic reticulum in cells. The coatomer of the vesicles coat is comprised of seven subunits: α-COP, ?-COP, β′-COP, β-COP, γ-COP, δ-COP, and ζ-COP. Here we report the solution structure of a truncated form (residues 1-149; ζ-COP149) of human ζ-COP (total 177 residues). It is the first three-dimensional structure of a “core” subunit of the COP I F-subcomplex. The structure of ζ-COP149 mainly consists of a disordered N-terminal tail, a five-stranded antiparallel β-sheet, a two-stranded antiparallel β-sheet, and five α-helices. The global folding of ζ-COP149 is very similar to the crystal structures of AP1-σ1 and AP2-σ2, directly demonstrating the structural similarity between the “core” subunits of the COP I F-subcomplex and adaptor protein complexes. Through structural comparison and mutagenesis study, we have also demonstrated that the heterodimers of ζ-COP149 and γ-COP have packing interfaces and relative subunit orientations similar to those of AP2-σ2 and AP2-α heterodimers. These results provide direct evidence supporting the previous proposal that the COP I F-subcomplex and adaptor protein complexes have similar tertiary and quaternary structures.     

Structure, folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli strains

Filamentous type 1 pili are responsible for attachment of uropathogenic Escherichia coli strains to host cells. They consist of a linear tip fibrillum and a helical rod formed by up to 3000 copies of the main structural pilus subunit FimA. The subunits in the pilus interact via donor strand complementation, where the incomplete, immunoglobulin-like fold of each subunit is complemented by an N-terminal donor strand of the subsequent subunit. Here, we show that folding of FimA occurs at an extremely slow rate (half-life: 1.6 h) and is catalyzed more than 400-fold by the pilus chaperone FimC. Moreover, FimA is capable of intramolecular self-complementation via its own donor strand, as evidenced by the loss of folding competence upon donor strand deletion. Folded FimA is an assembly-incompetent monomer of low thermodynamic stability (− 10.1 kJ mol^− 1) that can be rescued for pilus assembly at 37 °C because FimC selectively pulls the fraction of unfolded FimA molecules from the FimA folding equilibrium and allows FimA refolding on its surface. Elongation of FimA at the C-terminus by its own donor strand generated a self-complemented variant (FimAa) with alternative folding possibilities that spontaneously adopts the more stable conformation (− 85.0 kJ mol^− 1) in which the C-terminal donor strand is inserted in the opposite orientation relative to that in FimA. The solved NMR structure of FimAa revealed extensive β-sheet hydrogen bonding between the FimA pilin domain and the C-terminal donor strand and provides the basis for reconstruction of an atomic model of the pilus rod.     

Structure of the Murine Unglycosylated IgG1 Fc Fragment

A prototypic IgG antibody can be divided into two major structural units: the antigen-binding fragment (Fab) and the Fc fragment that mediates effector functions. The IgG Fc fragment is a homodimer of the two C-terminal domains (C_H2 and C_H3) of the heavy chains. Characteristic of the Fc part is the presence of a sugar moiety at the inner face of the C_H2 domains. The structure of this complex branched oligosaccharide is generally resolved in crystal structures of Fc fragments due to numerous well-defined sugar-protein interactions and a small number of sugar-sugar interactions. This suggested that sugars play an important role in the structure of the Fc fragment. To address this question directly, we determined the crystal structure of the unglycosylated Fc fragment of the murine IgG1 MAK33. The structures of the C_H3 domains of the unglycosylated Fc fragment superimpose perfectly with the structure of the isolated MAK33 C_H3 domain. The unglycosylated C_H2 domains, in contrast, approach each other much more closely compared to known structures of partly deglycosylated Fc fragments with rigid-body motions between 10 and 14 Å, leading to a strongly “closed” conformation of the unglycosylated Fc fragment. The glycosylation sites in the C′E loop and the BC and FG loops are well defined in the unglycosylated C_H2 domain, however, with increased mobility and with a significant displacement of about 4.9 Å for the unglycosylated Asn residue compared to the glycosylated structure. Thus, glycosylation both stabilizes the C′E-loop conformation within the C_H2 domain and also helps to ensure an “open” conformation, as seen upon Fc receptor binding. These structural data provide a rationale for the observation that deglycosylation of antibodies often compromises their ability to bind and activate Fcγ receptors.     

NMR Structure of the SARS-CoV Nonstructural Protein 7 in Solution at pH 6.5

The NMR structure of the severe acute respiratory syndrome coronavirus nonstructural protein (nsp) 7 in aqueous solution at pH 6.5 was determined and compared with the results of previous structure determinations of nsp7 in solution at pH 7.5 and in the crystals of a hexadecameric nsp7/nsp8 complex obtained from a solution at pH 7.5. All three structures contain four helices as the only regular secondary structures, but there are differences in the lengths and sequence locations of the four helices, as well as between the tertiary folds. The present study includes data on conformational equilibria and intramolecular rate processes in nsp7 in solution at pH 6.5, which provide further insights into the polymorphisms implicated by a comparison of the three presently available nsp7 structures.     

Probing possible downhill folding: native contact topology likely places a significant constraint on the folding cooperativity of proteins with approximately 40 residues

Experiments point to appreciable variations in folding cooperativity among natural proteins with approximately 40 residues, indicating that the behaviors of these proteins are valuable for delineating the contributing factors to cooperative folding. To explore the role of native topology in a protein's propensity to fold cooperatively and how native topology might constrain the degree of cooperativity achievable by a given set of physical interactions, we compared folding/unfolding kinetics simulated using three classes of native-centric C^α chain models with different interaction schemes. The approach was applied to two homologous 45-residue fragments from the peripheral subunit-binding domain family and a 39-residue fragment of the N-terminal domain of ribosomal protein L9. Free-energy profiles as functions of native contact number were computed to assess the heights of thermodynamic barriers to folding. In addition, chevron plots of folding/unfolding rates were constructed as functions of native stability to facilitate comparison with available experimental data. Although common Gō-like models with pairwise Lennard-Jones-type interactions generally fold less cooperatively than real proteins, the rank ordering of cooperativity predicted by these models is consistent with experiment for the proteins investigated, showing increasing folding cooperativity with increasing nonlocality of a protein's native contacts. Models that account for water-expulsion (desolvation) barriers and models with many-body (nonadditive) interactions generally entail higher degrees of folding cooperativity indicated by more linear model chevron plots, but the rank ordering of cooperativity remains unchanged. A robust, experimentally valid rank ordering of model folding cooperativity independent of the multiple native-centric interaction schemes tested here argues that native topology places significant constraints on how cooperatively a protein can fold.     

Conformational Dynamics and Structural Plasticity Play Critical Roles in the Ubiquitin Recognition of a UIM Domain

Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20-residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of interactions between UIMs and ubiquitin have shown that UIMs adopt an extended structure of a single α-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and the roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, we have characterized changes in the structure and dynamics of ubiquitin upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface, as well as global changes in ubiquitin due to UIM binding at the picoseconds-to-nanoseconds and microseconds-to-milliseconds protein motions by nuclear magnetic resonance relaxation. Changes in generalized-order parameters of amide groups show a distinct trend towards increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of ¹⁵N Carr-Purcell-Meiboom-Gill relaxation dispersion measurements suggests the presence of two types of motions: one directly related to the UIM-binding interface and the other induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects on protein motions at timescales ranging from picoseconds to milliseconds.