Folding of β-Hairpins

Identification of the factors governing the formation of -structure independently of the rest of the protein is important for understanding the folding process of protein into a unique native structure. It has been shown that some -hairpins can fold autonomously into native-like structures, either in aqueous solution or in the presence of an organic co-solvent. Our aim is to review recent theoretical and experimental studies of folding of -structures.     

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.     

Repositioning of Transmembrane α-Helices during Membrane Protein Folding

We have determined the optimal placement of individual transmembrane helices in the Pyrococcus horikoshii Glt_Ph glutamate transporter homolog in the membrane. The results are in close agreement with theoretical predictions based on hydrophobicity, but do not, in general, match the known three-dimensional structure, suggesting that transmembrane helices can be repositioned relative to the membrane during folding and oligomerization. Theoretical analysis of a database of membrane protein structures provides additional support for this idea. These observations raise new challenges for the structure prediction of membrane proteins and suggest that the classical two-stage model often used to describe membrane protein folding needs to be modified.     

Effects of Turn Stability and Side-Chain Hydrophobicity on the Folding of β-Structures

Key elements of β-structure folding include hydrophobic core collapse, turn formation, and assembly of backbone hydrogen bonds. In the present folding simulations of several β-hairpins and β-sheets (peptide 1, protein G B1 domain peptide, TRPZIP2, TRPZIP4, 20mer, and 20mer^DP6D), the folding free-energy landscape as a function of several reaction coordinates corresponding to the three key elements indicates apparent dependence on turn stability and side-chain hydrophobicity, which demonstrates different folding mechanisms of similar β-structures of varied sequences. Turn stability is found to be the key factor in determining the formation order of the three structural elements in the folding of β-structures. Moreover, turn stability and side-chain hydrophobicity both affect the stability of backbone hydrogen bonds. The three-stranded β-sheets fold through a three-state transition in which the formation of one hairpin always takes precedence over the other. The different stabilities of two anti-parallel hairpins in each three-stranded β-sheet are shown to correlate well with the different levels of their hydrophobic interactions.     

Folding up and Moving on—Nascent Protein Folding on the Ribosome

All cellular proteins are synthesized by the ribosome, an intricate molecular machine that translates the information of protein coding genes into the amino acid alphabet. The linear polypeptides synthesized by the ribosome must generally fold into specific three-dimensional structures to become biologically active. Folding has long been recognized to begin before synthesis is complete. Recently, biochemical and biophysical studies have shed light onto how the ribosome shapes the folding pathways of nascent proteins. Here, we discuss recent progress that is beginning to define the role of the ribosome in the folding of newly synthesized polypeptides.     

A Survey of λ Repressor Fragments from Two-State to Downhill Folding

We survey the two-state to downhill folding transition by examining 20 λ_6-85^? mutants that cover a wide range of stabilities and folding rates. We investigated four new λ_6-85^? mutants designed to fold especially rapidly. Two were engineered using the core remodeling of Lim and Sauer, and two were engineered using Ferreiro et al.'s frustratometer. These proteins have probe-dependent melting temperatures as high as 80 °C and exhibit a fast molecular phase with the characteristic temperature dependence of the amplitude expected for downhill folding. The survey reveals a correlation between melting temperature and downhill folding previously observed for the β-sheet protein WW domain. A simple model explains this correlation and predicts the melting temperature at which downhill folding becomes possible. An X-ray crystal structure with a 1.64-Å resolution of a fast-folding mutant fragment shows regions of enhanced rigidity compared to the full wild-type protein.     

Early Folding Events Protect Aggregation-Prone Regions of a β-Rich Protein

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Highlights? The folding pathway of a β-barrel protein protects it from aggregation ? The rate-determining transition state of CRABP1 is polarized and malleable ? Regions constituting the aggregate core of CRABP1 are protected early in folding ? Early barrel closure in iLBPs may offer a general strategy for productive folding     

Residue-Specific Analysis of Frustration in the Folding Landscape of Repeat β/α Protein Apoflavodoxin

Flavodoxin adopts the common repeat β/α topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central β-strand 3 (i.e., at β-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, ?-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the β-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central β-sheet directs initial misfolding, while proper alignment on both sides of β-strand 3 is necessary for productive folding.     

Why Do Protein Folding Rates Correlate with Metrics of Native Topology?

For almost 15 years, the experimental correlation between protein folding rates and the contact order parameter has been under scrutiny. Here, we use a simple simulation model combined with a native-centric interaction potential to investigate the physical roots of this empirical observation. We simulate a large set of circular permutants, thus eliminating dependencies of the folding rate on other protein properties (e.g. stability). We show that the rate-contact order correlation is a consequence of the fact that, in high contact order structures, the contact order of the transition state ensemble closely mirrors the contact order of the native state. This happens because, in these structures, the native topology is represented in the transition state through the formation of a network of tertiary interactions that are distinctively long-ranged.     

Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.Psychrophilic enzymes possess specific properties, such as high activity at low temperatures and weak thermal stability, which promise to allow the use of these enzymes as industrial biocatalysts, as biotechnological tools, or for fundamental research (6, 8, 11). For instance, substantial energy savings can be obtained if heating is not required during large-scale processes which take advantage of the efficient catalytic capacity of cold-adapted enzymes in the range 0 to 20°C. The pronounced heat lability of psychrophilic enzymes also allows their selective inactivation in a complex mixture, as illustrated by an antarctic bacterial alkaline phosphatase which is available for molecular biology research (7). Finally, psychrophilic enzymes represent the lower natural limit of protein stability (3) and are useful tools for studies in the field of protein folding.Large-scale fermentation of psychrophilic microorganisms suffers from two main drawbacks, however: the low production levels of wild strains and the prohibitive cost of growing wild strains at low temperatures. A possible alternative is to overexpress the gene coding for a psychrophilic protein in a mesophilic host for which efficient expression systems have been designed. In this context, two crucial questions remain to be solved: (i) what is the folding state of an enzyme normally synthesized at 0°C when it is expressed by the mesophilic genetic machinery at higher temperatures, and (ii) is there a temperature at which a compromise can be reached between the stability of the psychrophilic enzyme and the mesophilic growth rate? To address these questions, the heat-labile α-amylase from the antarctic psychrophile Alteromonas haloplanktis (2, 4) was expressed in Escherichia coli at various temperatures.

Construction of the expression vector and α-amylase production.

The α-amylase gene (2) was cloned downstream from the lacZ promoter in pUC12 by ligating the SmaI site of the polylinker to the HpaI site located 60 nucleotides upstream from the formylmethionine codon. This construction is devoid of the C-terminal peptide cleaved by the wild strain following α-amylase secretion. The recombinant enzyme was expressed in E. coli RR1 with the constitutive assistance of lacZ (without IPTG [isopropyl-β-d-thiogalactopyranoside] induction) in a medium containing 16 g of bactotryptone, 16 g of yeast extract, 5 g of NaCl, 2.5 g of K₂HPO₄, 0.1 μM CaCl₂, and 100 mg of ampicillin per liter. The effect of the culture temperature on α-amylase production by E. coli is illustrated in Fig. ​Fig.1.1. Within the range of temperatures used, maximal enzyme production was reached below 18°C, whereas higher temperatures induced a gradual decrease of α-amylase activity in cultures. Three independent cultures were pooled for the purification of the recombinant enzymes produced at 18 and 25°C. Open in a separate windowFIG. 1Temperature dependence of α-amylase production by E. coli. Results are expressed as percent mean maximal activity recorded at 18°C.

α-Amylase purification.

The gram-negative A. haloplanktis was cultivated at 4°C, and α-amylase was purified from the culture supernatants by ion-exchange chromatography on DEAE-agarose followed by gel filtration on Sephadex G-100 and Ultrogel AcA54 as previously described (2, 4). The recombinant α-amylases were purified by the protocol developed for the wild-type enzyme except that concentration by ammonium sulfate precipitation at 70% saturation was required before the first chromatographic step. Recombinant enzyme production at 18 and 25°C ranged between 60 and 100 mg/liter of culture, which corresponds to a 10-fold improvement over production by the wild strain.

Characterization of the recombinant α-amylases.

N- and C-terminal amino acid sequences (determined on an Applied Biosystems Procise analyzer and by carboxypeptidase Y digestion, respectively) of α-amylase produced at 18 and 25°C indicated that the signal peptide is correctly cleaved in E. coli and that no additional posttranslational cleavage occurred. The isoelectric point (5.5) and the molecular mass (49,340 Da as determined from the sequence and 49,342 ± 8 Da as determined from electrospray mass spectroscopy measurements) were identical to the values recorded for the wild-type enzyme. Dynamic light scattering (DynaPro-801; DLS Instruments) also showed that the purified recombinant enzymes are homogeneous, without any evidence of aggregated forms.

Comparison of the wild-type and recombinant α-amylases.

Several properties of the wild-type enzyme produced at 4°C and the recombinant α-amylase expressed in E. coli at 18°C were compared (Table ​(Table1).1).

TABLE 1

Kinetic parameters, dissociation constants, and free thiol groups for the wild-type and recombinant α-amylases

Understanding the Role of Three-Dimensional Topology in Determining the?Folding Intermediates of Group I Introns

Many RNA molecules exert their biological function only after folding to unique three-dimensional structures. For long, noncoding RNA molecules, the complexity of finding the native topology can be a major impediment to correct folding to the biologically active structure. An RNA molecule may fold to a near-native structure but not be able to continue to the correct structure due to a topological barrier such as crossed strands or incorrectly stacked helices. Achieving the native conformation thus requires unfolding and refolding, resulting in a long-lived intermediate. We investigate the role of topology in the folding of two phylogenetically related catalytic group I introns, the Twort and Azoarcus group I ribozymes. The kinetic models describing the Mg²⁺-mediated folding of these ribozymes were previously determined by time-resolved hydroxyl (⋅OH) radical footprinting. Two intermediates formed by parallel intermediates were resolved for each RNA. These data and analytical ultracentrifugation compaction analyses are used herein to constrain coarse-grained models of these folding intermediates as we investigate the role of nonnative topology in dictating the lifetime of the intermediates. Starting from an ensemble of unfolded conformations, we folded the RNA molecules by progressively adding native constraints to subdomains of the RNA defined by the ⋅OH time-progress curves to simulate folding through the different kinetic pathways. We find that nonnative topologies (arrangement of helices) occur frequently in the folding simulations despite using only native constraints to drive the reaction, and that the initial conformation, rather than the folding pathway, is the major determinant of whether the RNA adopts nonnative topology during folding. From these analyses we conclude that biases in the initial conformation likely determine the relative flux through parallel RNA folding pathways.     

Folding kinetics of the cooperatively folded subdomain of the IκBα ankyrin repeat domain

The ankyrin repeat (AR) domain of IκBα consists of a cooperative folding unit of roughly four ARs (AR1-AR4) and of two weakly folded repeats (AR5 and AR6). The kinetic folding mechanism of the cooperative subdomain, IκBα_67-206, was analyzed using rapid mixing techniques. Despite its apparent architectural simplicity, IκBα_67-206 displays complex folding kinetics, with two sequential on-pathway high-energy intermediates. The effect of mutations to or away from the consensus sequences of ARs on folding behavior was analyzed, particularly the GXTPLHLA motif, which have not been examined in detail previously. Mutations toward the consensus generally resulted in an increase in folding stability, whereas mutations away from the consensus resulted in decreased overall stability. We determined the free energy change upon mutation for three sequential transition state ensembles along the folding route for 16 mutants. We show that folding initiates with the formation of the interface of the outer helices of AR3 and AR4, and then proceeds to consolidate structure in these repeats. Subsequently, AR1 and AR2 fold in a concerted way in a single kinetic step. We show that this mechanism is robust to the presence of AR5 and AR6 as they do not strongly affect the folding kinetics. Overall, the protein appears to fold on a rather smooth energy landscape, where the folding mechanism conforms a one-dimensional approximation. However, we note that the AR does not necessarily act as a single folding element.     

Quantification of Excluded Volume Effects on the Folding Landscape of Pseudomonas aeruginosa Apoazurin In?Vitro

Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.     

The Effect of C-Terminal Deletion on the Folding and Self-association of Recombinant Non-phosphorylated Human ß-Casein

Recombinant human ß-casein (CN) mutants were prepared having 11, 22 and 31 amino acids (aa) deleted from the C-terminus. The temperature-dependent self-association of these and the wild-type recombinant was studied by turbidity (OD₄₀₀) while possible folding differences were examined by intrinsic and extrinsic fluorescence intensity and fluorescence resonance energy transfer. There were major self-association and some conformational differences. Hydrophobicity profile and hydrophobic cluster analysis for bovine and human ß-CN suggested that the ability of the 31 aa deletion mutant in human ß-CN to self-associate when a comparable bovine deletion peptide would not may be due to the presence of additional hydrophobic regions in the middle, indicating that the human protein may contain more than a single hydrophobic binding locus and suggesting that the process for the formation and structure of the micelles of human milk may be quite different from that for bovine milk. A new model may be needed.     

Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains

In eukaryotic cells, secretory pathway proteins must pass stringent quality control checkpoints before exiting the endoplasmic reticulum (ER). Acquisition of native structure is generally considered to be the most important prerequisite for ER exit. However, structurally detailed protein folding studies in the ER are few. Furthermore, aberrant ER quality control decisions are associated with a large and increasing number of human diseases, highlighting the need for more detailed studies on the molecular determinants that result in proteins being either secreted or retained. Here we used the clonotypic αβ chains of the T cell receptor (TCR) as a model to analyze lumenal determinants of ER quality control with a particular emphasis on how proper assembly of oligomeric proteins can be monitored in the ER. A combination of in vitro and in vivo approaches allowed us to provide a detailed model for αβTCR assembly control in the cell. We found that folding of the TCR α chain constant domain Cα is dependent on αβ heterodimerization. Furthermore, our data show that some variable regions associated with either chain can remain incompletely folded until chain pairing occurs. Together, these data argue for template-assisted folding at more than one point in the TCR α/β assembly process, which allows specific recognition of unassembled clonotypic chains by the ER chaperone machinery and, therefore, reliable quality control of this important immune receptor. Additionally, it highlights an unreported possible limitation in the α and β chain combinations that comprise the T cell repertoire.