A TeZla micromixer for interrogating the early and broad folding landscape of G-quadruplex via multistage velocity descending.

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures.

Differential effects of disulfide bond formation in TEM-1 versus CTX-M-9 ß-lactamase.

To investigate how disulfide bonds can impact protein energy landscapes, we surveyed the effects of adding or removing a disulfide in two ß-lactamase enzymes, TEM-1 and CTX-M-9. The homologs share a structure and 38% sequence identity, but only TEM-1 contains a native disulfide bond. They also differ in thermodynamic stability and in the number of states populated at equilibrium: CTX-M-9 is two-state whereas TEM-1 has an additional intermediate state. We hypothesized that the disulfide bond is the major underlying determinant for these observed differences in their energy landscapes. To test this, we removed the disulfide bridge from TEM-1 and introduced a disulfide bridge at the same location in CTX-M-9. This modest change to sequence modulates the stabilities-and therefore populations-of TEM-1's equilibrium states and, more surprisingly, creates a novel third state in CTX-M-9. Unlike TEM-1's partially folded intermediate, this third state is a higher-order oligomer with reduced cysteines that retains the native fold and is fully active. Sub-denaturing concentrations of urea shifts the equilibrium to the monomeric form, allowing the disulfide bond to form. Interestingly, comparing the stability of the oxidized monomer with a variant lacking cysteines reveals the disulfide is neither stabilizing nor destabilizing in CTX-M-9, in contrast with the observed stabilization in TEM-1. Thus, we can conclude that engineering disulfide bonds is not always an effective stabilization strategy even when analogous disulfides exist in more stable structural homologs. This study also illustrates how homo-oligomerization can result from a small number of mutations, suggesting complex formation might be easily accessed during a protein family's evolution.

Circular permutation at azurin's active site slows down its folding.

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn2+-bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn2+-CP protein was much slower than that of the Zn2+-WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn2+-azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn2+-cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn2+- WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes.

Non-Native Structures of Apomyoglobin and Apoleghemoglobin in Folding Intermediates Related to the Protein Misfolding.

Protein folding is essential for a polypeptide chain to acquire its proper structure and function. Globins are a superfamily of ubiquitous heme-binding α-helical proteins whose function is principally to regulate oxygen homoeostasis. In this review, we explore the hierarchical helical formation in the globin proteins apomyoglobin and leghemoglobin, and we discuss the existence of non-native and misfolded structures occurring during the course of folding to its native state. This review summarizes the research aimed at characterizing and comparing the equilibrium and kinetic intermediates, as well as delineating the complete folding pathway at a molecular level, in order to answer the following questions: "What is the mechanism of misfolding via a folding intermediate? Does the non-native structure stabilize the contemporary intermediate structure? Does the non-native structure induce slower folding?" The role of the non-native structures in the folding intermediate related to misfolding is also discussed.

A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins.

Investigations of protein folding have largely involved the use of disulfide-containing proteins, since the disulfide-coupled folding of proteins allows folding intermediates to be trapped and their conformations determined. However, studies of the folding mechanisms of mid-size proteins face several problems, one of which is that detecting folding intermediates is difficult. Therefore, to solve this issue, a novel peptide reagent, maleimidohexanoyl-Arg5-Tyr-NH2, was designed and applied to the detection of folding intermediates of model proteins. BPTI was chosen as a model small protein to estimate the ability of the novel reagent to detect folding intermediates. In addition, a precursor protein (prococoonase) of Bombyx mori cocoonase was used as a model mid-size protein. Cocoonase is classified as a serine protease and has a high homology with trypsin. We recently found that the propeptide sequence of prococoonase (proCCN) is important for the folding of cocoonase. However, it was difficult to study the folding pathway of proCCN since the folding intermediates could not be separated on a reversed-phase HPLC (RP-HPLC). Therefore, to separate the folding intermediates by RP-HPLC, the novel labeling reagent was used to accomplish this for proCCN. The results indicated that the peptide reagent allowed the intermediates to be captured, separated on SDS-PAGE, and analyzed by RP-HPLC without the occurrence of undesirable disulfide-exchange reactions during the labeling reactions. The peptide reagent reported herein is a practical tool for investigating the mechanisms of disulfide-coupled folding of mid-size proteins.

Nanopore single-molecule biosensor in protein denaturation analysis.

Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the "direct" model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.

Elucidation of folding pathways of knotted proteins.

Understanding the mechanisms by which proteins fold and thread into topologically knotted conformations has been challenging because of the apparent complexity associated with the folding and threading events. Nevertheless, many experimental and computational studies have provided insights into the folding pathways of knotted proteins and showed that most of the knotted proteins could spontaneously and reversibly fold into knotted topologies with highly populated intermediates and, at times, through multiple folding pathways. Our laboratory has reported the folding mechanisms of a variety of knotted proteins that have different knot types, ranging from the simplest trefoil 31 knot to the most complex Stevedore's 61 knot. Therefore, we focused on using multiplex thermodynamics and kinetics measurements to tease out unique information associated with different structural probes to obtain a more comprehensive overview of the folding mechanisms of the knotted proteins of interest. In this chapter, we shall discuss the use of different biophysical tools and analytical models to glean mechanistic insights into how intricate polypeptides attain knotted topologies.

Folding Mechanism and Aggregation Propensity of the KH0 Domain of FMRP and Its R138Q Pathological Variant.

The K-homology (KH) domains are small, structurally conserved domains found in proteins of different origins characterized by a central conserved ßααß "core" and a GxxG motif in the loop between the two helices of the KH core. In the eukaryotic KHI type, additional αß elements decorate the "core" at the C-terminus. Proteins containing KH domains perform different functions and several diseases have been associated with mutations in these domains, including those in the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein crucial for the control of RNA metabolism whose lack or mutations lead to fragile X syndrome (FXS). Among missense mutations, the R138Q substitution is in the KH0 degenerated domain lacking the classical GxxG motif. By combining equilibrium and kinetic experiments, we present a characterization of the folding mechanism of the KH0 domain from the FMRP wild-type and of the R138Q variant showing that in both cases the folding mechanism implies the accumulation of an on-pathway transient intermediate. Moreover, by exploiting a battery of biophysical techniques, we show that the KH0 domain has the propensity to form amyloid-like aggregates in mild conditions in vitro and that the R138Q mutation leads to a general destabilization of the protein and to an increased fibrillogenesis propensity.

Peptide Model of the Mutant Proinsulin Syndrome. II. Nascent Structure and Biological Implications.

Toxic misfolding of proinsulin variants in ß-cells defines a monogenic diabetes syndrome, designated mutant INS-gene induced diabetes of the young (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe 1H and 1H-13C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic 13C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains LeuB15, LeuA16 and PheB24 at the nexus of nativelike α-helices α1 and α3 (as defined in native proinsulin) and flanking ß-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > SerB24 >> ProA16 > ProB15 (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome's variable phenotypic spectrum-onsets ranging from the neonatal period to later in childhood or adolescence-reflects structural features of respective folding intermediates.

Peptide Model of the Mutant Proinsulin Syndrome. I. Design and Clinical Correlation.

The mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin's biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of ß-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin's first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin's putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides.

Structure-activity relationship for the folding intermediate-selective inhibition of DYRK1A.

DYRK1A phosphorylates proteins involved in neurological disorders in an intermolecular manner. Meanwhile, during the protein folding process of DYRK1A, a transitional folding intermediate catalyzes the intramolecular autophosphorylation required for the "one-off" inceptive activation and stabilization. In our previous study, a small molecule termed FINDY (1) was identified, which inhibits the folding intermediate-catalyzed intramolecular autophosphorylation of DYRK1A but not the folded state-catalyzed intermolecular phosphorylation. However, the structural features of FINDY (1) responsible for this intermediate-selective inhibition remain elusive. In this study, structural derivatives of FINDY (1) were designed and synthesized according to its predicted binding mode in the ATP pocket of DYRK1A. Quantitative structure-activity relationship (QSAR) of the derivatives revealed that the selectivity against the folding intermediate is determined by steric hindrance between the bulky hydrophobic moiety of the derivatives and the entrance to the pocket. In addition, a potent derivative 3 was identified, which inhibited the folding intermediate more strongly than FINDY (1); it was designated as dp-FINDY. Although dp-FINDY (3) did not inhibit the folded state, as well as FINDY (1), it inhibited the intramolecular autophosphorylation of DYRK1A in an in vitro cell-free protein synthesis assay. Furthermore, dp-FINDY (3) destabilized endogenous DYRK1A in HEK293 cells. This study provides structural insights into the folding intermediate-selective inhibition of DYRK1A and expands the chemical options for the design of a kinase inhibitor.

FtsH degrades dihydrofolate reductase by recognizing a partially folded species.

AAA+ proteolytic machines play essential roles in maintaining and rebalancing the cellular proteome in response to stress, developmental cues, and environmental changes. Of the five AAA+ proteases in Escherichia coli, FtsH is unique in its attachment to the inner membrane and its function in degrading both membrane and cytosolic proteins. E. coli dihydrofolate reductase (DHFR) is a stable and biophysically well-characterized protein, which a previous study found resisted FtsH degradation despite the presence of an ssrA degron. By contrast, we find that FtsH degrades DHFR fused to a long peptide linker and ssrA tag. Surprisingly, we also find that FtsH degrades DHFR with shorter linkers and ssrA tag, and without any linker or tag. Thus, FtsH must be able to recognize a sequence element or elements within DHFR. We find that FtsH degradation of DHFR is noncanonical in the sense that it does not rely upon recognition of an unstructured polypeptide at or near the N-terminus or C-terminus of the substrate. Results using peptide-array experiments, mutant DHFR proteins, and fusion proteins suggest that FtsH recognizes an internal sequence in a species of DHFR that is partially unfolded. Overall, our findings provide insight into substrate recognition by FtsH and indicate that its degradation capacity is broader than previously reported.

Folding and Misfolding of a PDZ Tandem Repeat.

Although the vast majority of the human proteome is represented by multi-domain proteins, the study of multi-domain folding and misfolding is a relatively poorly explored field. The protein Whirlin is a multi-domain scaffolding protein expressed in the inner ear. It is characterized by the presence of tandem repeats of PDZ domains. The first two PDZ domains of Whirlin (PDZ1 and PDZ2 - namely P1P2) are structurally close and separated by a disordered short linker. We recently described the folding mechanism of the P1P2 tandem. The difference in thermodynamic stability of the two domains allowed us to selectively unfold one or both PDZ domains and to pinpoint the accumulation of a misfolded intermediate, which we demonstrated to retain physiological binding activity. In this work, we provide an extensive characterization of the folding and unfolding of P1P2. Based on the observed data, we describe an integrated kinetic analysis that satisfactorily fits the experiments and provides a valuable model to interpret multi-domain folding. The experimental and analytical approaches described in this study may be of general interest for the interpretation of complex multi-domain protein folding kinetics.

Druggable Transient Pockets in Protein Kinases.

Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of "cryptic inhibitor-binding sites." These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.

The molecular species responsible for α1 -antitrypsin deficiency are suppressed by a small molecule chaperone.

The formation of ordered Z (Glu342Lys) α1 -antitrypsin polymers in hepatocytes is central to liver disease in α1 -antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation-selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse-chase experiments demonstrate that Z α1 -antitrypsin accumulates as short-chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half-life (10.9 ± 1.7 h and 20.9 ± 7.4 h for soluble and insoluble polymers, respectively). The M* intermediate (or a by-product thereof) was identified in the cells by a conformation-specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α1 -antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease.

Insight into the Folding and Dimerization Mechanisms of the N-Terminal Domain from Human TDP-43.

TAR DNA-binding protein 43 (TDP-43) is a 414-residue long nuclear protein whose deposition into intraneuronal insoluble inclusions has been associated with the onset of amyotrophic lateral sclerosis (ALS) and other diseases. This protein is physiologically a homodimer, and dimerization occurs through the N-terminal domain (NTD), with a mechanism on which a full consensus has not yet been reached. Furthermore, it has been proposed that this domain is able to affect the formation of higher molecular weight assemblies. Here, we purified this domain and carried out an unprecedented characterization of its folding/dimerization processes in solution. Exploiting a battery of biophysical approaches, ranging from FRET to folding kinetics, we identified a head-to-tail arrangement of the monomers within the dimer. We found that folding of NTD proceeds through the formation of a number of conformational states and two parallel pathways, while a subset of molecules refold slower, due to proline isomerism. The folded state appears to be inherently prone to form high molecular weight assemblies. Taken together, our results indicate that NTD is inherently plastic and prone to populate different conformations and dimeric/multimeric states, a structural feature that may enable this domain to control the assembly state of TDP-43.

Molecular dynamics of far positioned surface mutations of Cu/Zn SOD1 promotes altered structural stability and metal-binding site: Structural clues to the pathogenesis of amyotrophic lateral sclerosis.

Cu/Zn superoxide dismutase (SOD1) mutations are associated to the motor neuron disorder, amyotrophic lateral sclerosis (ALS), which is characterized by aggregates of the misfolded proteins. The distribution of mutations all over the three-dimensional structure of SOD1 makes it complex to determine the exact molecular mechanism underlying SOD1 destabilization and the associated ALS pathology. In this study, we have examined structure and dynamics of SOD1 protein upon two ALS associated point mutations at the surface residue Glu100 (E100G and E100K), which is located far from the Cu and Zn sites and dimer interface. The molecular dynamics simulations were performed for these mutants for 50ns using GROMACS package. Our results indicate that the mutations result in structural destabilization by affecting the gate keeping role of Glu100 and loss of electrostatic interactions on the protein surface which stabilizes the ß-barrel structure of the native form. Further, these mutations could increase the fluctuations in the zinc-binding loop (loop IV), primarily due to loss of hydrogen bond between Asp101 and Arg79. The relaxed conformation of Arg79 further affects the native conformation of His80 and Asp83, that results in altered zinc site geometry and the structure of the substrate channel. Our results clearly suggest that, similar to the mutations located at metal sites/dimer interface/disulfide regions, the mutations at the far positioned site (Glu100) also induce significant conformational changes that could affect the metallation and structure of SOD1 molecule, resulting in formation of toxic intermediate species that cause ALS.

The role of the IT-state in D76N ß2-microglobulin amyloid assembly: A crucial intermediate or an innocuous bystander?

The D76N variant of human ß2-microglobulin (ß2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of WT-ß2m, which occurs in dialysis-related amyloidosis. A folding intermediate of WT-ß2m, known as the IT-state, which contains a nonnative trans Pro-32, has been shown to be a key precursor of WT-ß2m aggregation in vitro However, how a single amino acid substitution enhances the rate of aggregation of D76N-ß2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-ß2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-ß2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analog of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-ß2m but slows aggregation of D76N-ß2m, supporting the view that although the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the origin of the rapid aggregation of D76N-ß2m, suggesting that other nonnative states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.

Hierarchical Conformational Dynamics Confers Thermal Adaptability to preQ1 RNA Riboswitches.

Single-stranded noncoding regulatory RNAs, as exemplified by bacterial riboswitches, are highly dynamic. The conformational dynamics allow the riboswitch to reach maximum switching efficiency under appropriate conditions. Here we characterize the conformational dynamics of preQ1 riboswitches from mesophilic and thermophilic bacterial species at various temperatures. With the integrative use of small-angle X-ray scattering, NMR, and molecular dynamics simulations, we model the ensemble-structures of the preQ1 riboswitch aptamers without or with a ligand bound. We show that the preQ1 riboswitch is sufficiently dynamic and fluctuating among multiple folding intermediates only near the physiological temperature of the microorganism. The hierarchical folding dynamics of the RNA involves the docking of 3'-tail to form a second RNA helix and the helical stacking to form an H-type pseudoknot structure. Further, we show that RNA secondary and tertiary dynamics can be modulated by temperature and by the length of an internal loop. The coupled equilibria between RNA folding intermediates are essential for preQ1 binding, and a four-state exchange model can account for the change of ligand-triggered switching efficiency with temperature. Together, we have established a relationship between the hierarchical dynamics and riboswitch function, and illustrated how the RNA adapts to high temperature.

Structure and Oxidative Folding of AAI, the Major Alfa-Amylase Inhibitor From Amaranth Seeds.

AAI, the major alpha-amylase inhibitor (AAI) present in the seeds of the Mexican crop plant Amaranthus hypocondriacus is a 32-residue-long polypeptide with three disulfide bridges. Its structure is most closely related to the plant amylase inhibitor subfamily of knottins characterized by a topological knot formed by one disulfide bridge threading through a loop formed by the peptide chain as well as a short three-stranded beta sandwich core. AAI is specific against insect amylases and does not act on corresponding human or mammalian enzymes. It was found that the oxidative folding of AAI seems to follow a hirudine-like pathway with many non-native intermediates, but notably it proceeds through a major folding intermediate (MFI) that contains a vicinal disulfide bridge. Based on a review of the pertinent literature, the known vicinal disulfides in native proteins as well as well as the network of disulfide interchanges, we propose that MFI is a kinetic trap corresponding to a compact molten globule-like state which constrains the peptide chain to a smaller number of conformations that in turn can be rapidly funneled toward the native state.