The Positively Charged Cluster in the N-terminal Disordered Region may Affect Prion Protein Misfolding: Cryo-EM Structure of Hamster PrP(23-144) Fibrils.

Prions, the misfolding form of prion proteins, are contagious proteinaceous macromolecules. Recent studies have shown that infectious prion fibrils formed in the brain and non-infectious fibrils formed from recombinant prion protein in a partially denaturing condition have distinct structures. The amyloid core of the in vitro-prepared non-infectious fibrils starts at about residue 160, while that of infectious prion fibrils formed in the brain involves a longer sequence (residues ∼90-230) of structural conversion. The C-terminal truncated prion protein PrP(23-144) can form infectious fibrils under certain conditions and cause disease in animals. In this study, we used cryogenic electron microscopy (cryo-EM) to resolve the structure of hamster sHaPrP(23-144) fibrils prepared at pH 3.7. This 2.88 Å cryo-EM structure has an amyloid core covering residues 94-144. It comprises two protofilaments, each containing five ß-strands arranged as a long hairpin plus an N-terminal ß-strand. This N-terminal ß-strand resides in a positively charged cluster region (named PCC2; sequence 96-111), which interacts with the turn region of the opposite protofilaments' hairpin to stabilize the fibril structure. Interestingly, this sHaPrP(23-144) fibril structure differs from a recently reported structure formed by the human or mouse counterpart at pH 6.5. Moreover, sHaPrP(23-144) fibrils have many structural features in common with infectious prions. Whether this structure is infectious remains to be determined. More importantly, the sHaPrP(23-144) structure is different from the sHaPrP(108-144) fibrils prepared in the same fibrillization buffer, indicating that the N-terminal disordered region, possibly the positively charged cluster, influences the misfolding pathway of the prion protein.

Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography.

The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

Cholesterol twists the transmembrane Di-Gly region of amyloid-precursor protein.

Nearly 95% of Alzheimer's disease (AD) occurs sporadically without genetic linkage. Aging, hypertension, high cholesterol content, and diabetes are known nongenomic risk factors of AD. Aggregation of Aß peptides is an initial event of AD pathogenesis. Aß peptides are catabolic products of a type I membrane protein called amyloid precursor protein (APP). Aß40 is the major product, whereas the 2-residue-longer version, Aß42, induces amyloid plaque formation in the AD brain. Since cholesterol content is one risk factor for sporadic AD, we aimed to explore whether cholesterol in the membrane affects the structure of the APP transmembrane region, thereby modulating the γ-secretase cutting behavior. Here, we synthesized several peptides containing the APP transmembrane region (sequence 693-726, corresponding to the Aß22-55 sequence) with one or two Cys mutations for spin labeling. We performed three electron spin resonance experiments to examine the structural changes of the peptides in liposomes composed of dioleoyl phosphatidylcholine and different cholesterol content. Our results show that cholesterol increases membrane thickness by 10% and peptide length accordingly. We identified that the di-glycine region of Aß36-40 (sequence VGGVV) exhibits the most profound change in response to cholesterol compared with other segments, explaining how the presence of cholesterol affects the γ-secretase cutting site. This study provides spectroscopic evidence showing how cholesterol modulates the structure of the APP transmembrane region in a lipid bilayer.

Cross-Seeding Assay in the Investigation of the Amyloid Core of Prion Fibrils.

Amyloidogenesis, self-propagation of protein or peptide monomers to amyloid fibrils, has been linked to incurable pathogenesis of neurodegenerative diseases such as Alzheimer's disease and prion diseases. Investigations of amyloid structures and how monomers are transformed through seeding are therefore crucial for developing therapeutics toward these diseases. Here we describe a cross-seeding method to explore the amyloid core in prion fibrils that uses preformed amyloid fibrils as a seed to induce the transformation of other protein or peptide monomers to amyloid fibrils.

2.2 Å Cryo-EM Tetra-Protofilament Structure of the Hamster Prion 108-144 Fibril Reveals an Ordered Water Channel in the Center.

Fibrils of the hamster prion peptide (sHaPrP, sequence 108-144) were prepared in an acidic solution, and their structure was solved by cryogenic electron microscopy with a resolution of 2.23 Å based on the gold-standard Fourier shell correlation (FSC) curve. The fibril has a novel architecture that has never been found in other amyloid fibrils. Each fibril is assembled by four protofilaments (PFs) and has an ordered water channel in the center. Each protofilament contains three ß-strands (125-130, 133-135, and 138-141) arranged in an "R"-shaped construct. The structural data indicate that these three ß-strand segments are the most amyloidogenic region of the prion peptide/protein and might be the site of nucleation during fibrillization under conditions without denaturants.

Location of the cross-ß structure in prion fibrils: A search by seeding and electron spin resonance spectroscopy.

Prion diseases are transmissible fatal neurodegenerative disorders spreading between humans and other mammals. The pathogenic agent, prion, is a protease-resistant, ß-sheet-rich protein aggregate, converted from a membrane protein called PrPC . PrPSc is the misfolded form of PrPC and undergoes self-propagation to form the infectious amyloids. Since the key hallmark of prion disease is amyloid formation, identifying and studying which segments are involved in the amyloid core can provide molecular details about prion diseases. It has been known that the prion protein could also form non-infectious fibrils in the presence of denaturants. In this study, we employed a combination of site-directed nitroxide spin-labeling, fibril seeding, and electron spin resonance (ESR) spectroscopy to identify the structure of the in vitro-prepared full-length mouse prion fibrils. It is shown that in the in vitro amyloidogenesis, the formation of the amyloid core is linked to an α-to-ß structural transformation involving the segment 160-224, which contains strand 2, helix 2, and helix 3. This method is particularly suitable for examining the hetero-seeded amyloid fibril structure, as the unlabeled seeds are invisible by ESR spectroscopy. It can be applied to study the structures of different strains of infectious prions or other amyloid fibrils in the future.

Condition-dependent structural collapse in the intrinsically disordered N-terminal domain of prion protein.

Prion protein is composed of a structure-unsolved N-terminal domain and a globular C-terminal domain. Under limited trypsin digestion, mouse recombinant prion protein can be cleaved into two parts at residue Lys105. Here, we termed these two fragments as the N-domain (sequence 23-105) and the C-domain (sequence 106-230). In this study, the structural properties of the N-domain, the C-domain, and the full-length protein were explored using small-angle X-ray scattering, analytical ultracentrifugation, circular dichroism spectroscopy, and the 8-anilino-1-naphthalenesulfonic acid binding assay. The conformation and size of the prion protein were found to change sensitively under the solvent conditions. The positive residues in the sequence 23-99 of the N-domain were found to be responsible for the enhanced flexibility with the salt concentration reduced below 5 mM. The C-domain containing a hydrophobic patch tends to unfold and aggregate during a salt-induced structural collapse. The N-domain collapsed together with the C-domain at pH 5.2, whereas it collapsed independently at pH 4.2. The positively charged cluster (sequence 100-105) in the N-domain contributed to protecting the exposed hydrophobic surface of the C-domain.

De Novo Design of Antimicrobial Peptides With a Special Charge Pattern and Their Application in Combating Plant Pathogens.

Plant diseases are important issues in agriculture, and the development of effective and environment-friendly means of disease control is crucial and highly desired. Antimicrobial peptides (AMPs) are known as potential alternatives to chemical pesticides because of their potent broad-spectrum antimicrobial activity and because they have no risk, or have only a low risk, of developing chemical-resistant pathogens. In this study, we designed a series of amphipathic helical peptides with different spatial distributions of positive charges and found that the peptides that had a special sequence pattern "BBHBBHHBBH" ("B" for basic residue and "H" for hydrophobic residue) displayed excellent bactericidal and fungicidal activities in a wide range of economically important plant pathogens. The peptides with higher helical propensity had lower antimicrobial activity. When we modified the peptides with a long acyl chain at their N-terminus, their plant protection effect improved. Our application of the fatty acyl-modified peptides on the leaves of tomato and Arabidopsis plants lessened the infection caused by Pectobacterium carotovorum subsp. carotovorum and Botrytis cinerea. Our study provides important insights on the development of more potent novel AMPs for plant protection.

Synthesis and Biological Evaluation of Hydroxylated Monocarbonyl Curcumin Derivatives as Potential Inducers of Neprilysin Activity.

BACKGROUND: Alzheimer's disease (AD) involves impairment of Aß clearance. Neprilysin (NEP) is the most efficient Aß peptidase. Enhancement of the activity or expression of NEP may provide a prominent therapeutic strategy against AD. AIMS: Ten hydroxylated monocarbonyl curcumin derivatives were designed, synthesized and evaluated for their NEP upregulating potential using sensitive fluorescence-based Aß digestion and inhibition assays. RESULTS: Compound 4 was the most active one, resulting in a 50% increase in Aß cleavage activity. Cyclohexanone-bearing derivatives exhibited higher activity enhancement compared to their acetone counterparts. Inhibition experiments with the NEP-specific inhibitor thiorphan resulted in dramatic cleavage reduction. Conclusion: The increased Aß cleavage activity and the ease of synthesis of 4 renders it an extremely attractive lead compound.

A Systematic Study of the Stability, Safety, and Efficacy of the de novo Designed Antimicrobial Peptide PepD2 and Its Modified Derivatives Against Acinetobacter baumannii.

Searching for new antimicrobials is a pressing issue to conquer the emergence of multidrug-resistant (MDR) bacteria and fungi. Antimicrobial peptides (AMPs) usually have antimicrobial mechanisms different from those of traditional antibiotics and bring new hope in the discovery of new antimicrobials. In addition to antimicrobial activity, stability and target selectivity are important concerns to decide whether an antimicrobial peptide can be applied in vivo. Here, we used a simple de novo designed peptide, pepD2, which contains only three kinds of amino acid residues (W, K, L), as an example to evaluate how the residues and modifications affect the antimicrobial activity against Acinetobacter baumannii, stability in plasma, and toxicity to human HEK293 cells. We found that pepI2 with a Leu→Ile substitution can decrease the minimum bactericidal concentrations (MBC) against A. baumannii by one half (4 µg/mL). A D-form peptide, pepdD2, in which the D-enantiomers replaced the L-enantiomers of the Lys(K) and Leu(L) residues, extended the peptide half-life in plasma by more than 12-fold. PepD3 is 3-residue shorter than pepD2. Decreasing peptide length did not affect antimicrobial activity but increased the IC50 to HEK293 cells, thus increased the selectivity index (SI) between A. baumannii and HEK293 cells from 4.7 to 8.5. The chain length increase of the N-terminal acyl group and the Lys→Arg substitution greatly enhanced the hemolytic activity, hence those modifications are not good for clinical application. Unlike colistin, the action mechanism of our peptides relies on negatively charged lipids rather than lipopolysaccharides. Therefore, not only gram-negative bacteria but also gram-positive bacteria can be killed by our peptides.

Probiotic Bacillus Affects Enterococcus faecalis Antibiotic Resistance Transfer by Interfering with Pheromone Signaling Cascades.

Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis subsp. natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from chromosomally encoded CF10 (cCF10) without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30- to 50-kDa extracellular protease present in B. subtilis subsp. natto. Nattokinase of B. subtilis subsp. natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into LVTL plus VFV fragments. Moreover, the cleavage product LVTL (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis subsp. natto can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis subsp. natto and L peptide to mitigate E. faecalis antibiotic resistance transfer. IMPORTANCE Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, antibiotic resistance genes can undergo efficient intra- and interspecies transfer via E. faecalis peptide pheromone-mediated plasmid transfer systems. Therefore, this study provided the first experimental demonstration that probiotics are a feasible approach for interfering with conjugative plasmid transfer between E. faecalis strains to stop the transfer of antibiotic resistance. We found that the extracellular protease(s) of Bacillus subtilis subsp. natto cleaved peptide pheromones without affecting the growth of E. faecalis, thereby reducing the frequency of conjugative plasmid transfer. In addition, a specific cleaved pheromone fragment interfered with conjugative plasmid transfer. These findings provide a potential probiotic-based method for interfering with the transfer of antibiotic resistance between E. faecalis strains.

Corrigendum: A Systematic Study of the Stability, Safety, and Efficacy of the de novo Designed Antimicrobial Peptide PepD2 and Its Modified Derivatives Against Acinetobacter baumannii.

[This corrects the article DOI: 10.3389/fmicb.2021.678330.].

Self-Replication of Prion Protein Fragment 89-230 Amyloid Fibrils Accelerated by Prion Protein Fragment 107-143 Aggregates.

Prion protein amyloid aggregates are associated with infectious neurodegenerative diseases, known as transmissible spongiform encephalopathies. Self-replication of amyloid structures by refolding of native protein molecules is the probable mechanism of disease transmission. Amyloid fibril formation and self-replication can be affected by many different factors, including other amyloid proteins and peptides. Mouse prion protein fragments 107-143 (PrP(107-143)) and 89-230 (PrP(89-230)) can form amyloid fibrils. ß-sheet core in PrP(89-230) amyloid fibrils is limited to residues ∼160-220 with unstructured N-terminus. We employed chemical kinetics tools, atomic force microscopy and Fourier-transform infrared spectroscopy, to investigate the effects of mouse prion protein fragment 107-143 fibrils on the aggregation of PrP(89-230). The data suggest that amyloid aggregates of a short prion-derived peptide are not able to seed PrP(89-230) aggregation; however, they accelerate the self-replication of PrP(89-230) amyloid fibrils. We conclude that PrP(107-143) fibrils could facilitate the self-replication of PrP(89-230) amyloid fibrils in several possible ways, and that this process deserves more attention as it may play an important role in amyloid propagation.

Segments in the Amyloid Core that Distinguish Hamster from Mouse Prion Fibrils.

Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and other mammals. The disease transmission can occur between different species but is limited by the sequence homology between host and inoculum. The crucial molecular event in the progression of this disease is prion formation, starting from the conformational conversion of the normal, membrane-anchored prion protein (PrPC) into the misfolded, ß-sheet-rich and aggregation-prone isoform (PrPSc), which then self-associates into the infectious amyloid form called prion. Amyloid is the aggregate formed from one-dimensional protein association. As amyloid formation is a key hallmark in prion pathogenesis, studying which segments in prion protein are involved in the amyloid formation can provide molecular details in the cross-species transmission barrier of prion diseases. However, due to the difficulties of studying protein aggregates, very limited knowledge about prion structure or prion formation was disclosed by now. In this study, cross-seeding assay was used to identify the segments involved in the amyloid fibril formation of full-length hamster prion protein, SHaPrP(23-231). Our results showed that the residues in the segments 108-127, 172-194 (helix 2 in PrPC) and 200-227 (helix 3 in PrPC) are in the amyloid core of hamster prion fibrils. The segment 127-143, but not 107-126 (which corresponds to hamster sequence 108-127), was previously reported to be involved in the amyloid core of full-length mouse prion fibrils. Our results indicate that hamster prion protein and mouse prion protein use different segments to form the amyloid core in amyloidogenesis. The sequence-dependent core formation can be used to explain the seeding barrier between mouse and hamster.

Directly monitor protein rearrangement on a nanosecond-to-millisecond time-scale.

In order to directly observe the refolding kinetics from a partially misfolded state to a native state in the bottom of the protein-folding funnel, we used a "caging" strategy to trap the ß-sheet structure of ubiquitin in a misfolded conformation. We used molecular dynamics simulation to generate the cage-induced, misfolded structure and compared the structure of the misfolded ubiquitin with native ubiquitin. Using laser flash irradiation, the cage can be cleaved from the misfolded structure within one nanosecond, and we monitored the refolding kinetics of ubiquitin from this misfolded state to the native state by photoacoustic calorimetry and photothermal beam deflection techniques on nanosecond to millisecond timescales. Our results showed two refolding events in this refolding process. The fast event is shorter than 20 ns and corresponds to the instant collapse of ubiquitin upon cage release initiated by laser irradiation. The slow event is ~60 µs, derived from a structural rearrangement in ß-sheet refolding. The event lasts 10 times longer than the timescale of ß-hairpin formation for short peptides as monitored by temperature jump, suggesting that rearrangement of a ß-sheet structure from a misfolded state to its native state requires more time than ab initio folding of a ß-sheet.

An intranasally delivered peptide drug ameliorates cognitive decline in Alzheimer transgenic mice.

Alzheimer's disease (AD) is the most common neurodegenerative disease. Imbalance between the production and clearance of amyloid ß (Aß) peptides is considered to be the primary mechanism of AD pathogenesis. This amyloid hypothesis is supported by the recent success of the human anti-amyloid antibody aducanumab, in clearing plaque and slowing clinical impairment in prodromal or mild patients in a phase Ib trial. Here, a peptide combining polyarginines (polyR) (for charge repulsion) and a segment derived from the core region of Aß amyloid (for sequence recognition) was designed. The efficacy of the designed peptide, R8-Aß(25-35), on amyloid reduction and the improvement of cognitive functions were evaluated using APP/PS1 double transgenic mice. Daily intranasal administration of PEI-conjugated R8-Aß(25-35) peptide significantly reduced Aß amyloid accumulation and ameliorated the memory deficits of the transgenic mice. Intranasal administration is a feasible route for peptide delivery. The modular design combining polyR and aggregate-forming segments produced a desirable therapeutic effect and could be easily adopted to design therapeutic peptides for other proteinaceous aggregate-associated diseases.

ErbB2 regulates autophagic flux to modulate the proteostasis of APP-CTFs in Alzheimer's disease.

Proteolytic processing of amyloid precursor protein (APP) C-terminal fragments (CTFs) by γ-secretase underlies the pathogenesis of Alzheimer's disease (AD). An RNA interference screen using APP-CTF [99-residue CTF (C99)]- and Notch-specific γ-secretase interaction assays identified a unique ErbB2-centered signaling network that was predicted to preferentially govern the proteostasis of APP-C99. Consistently, significantly elevated levels of ErbB2 were confirmed in the hippocampus of human AD brains. We then found that ErbB2 effectively suppressed autophagic flux by physically dissociating Beclin-1 from the Vps34-Vps15 complex independent of its kinase activity. Down-regulation of ErbB2 by CL-387,785 decreased the levels of C99 and secreted amyloid-ß in cellular, zebrafish, and mouse models of AD, through the activation of autophagy. Oral administration of an ErbB2-targeted CL-387,785 for 3 wk significantly improves the cognitive functions of APP/presenilin-1 (PS1) transgenic mice. This work unveils a noncanonical function of ErbB2 in modulating autophagy and establishes ErbB2 as a therapeutic target for AD.

Examining the inhibitory potency of food additive fast green FCF against amyloid fibrillogenesis under acidic conditions.

More than thirty human proteins and/or peptides can fold incorrectly to form amyloid deposits associated with several protein aggregation diseases. No cure is currently available for treating these diseases. This work is aimed at examining the inhibitory potency of fast green FCF, a biocompatible dye, toward the fibrillogenesis/aggregation of lysozyme. As verified by ThT binding assay along with transmission electron microscopy, fast green FCF was observed to suppress the generation of lysozyme fibrils in a concentration-dependent manner. We next used circular dichroism absorption spectroscopy, ANS fluorescence spectroscopy, and SDS-PAGE to characterize the structural alterations in lysozyme samples upon the addition of fast green FCF. Furthermore, experiments with the addition of fast green FCF at different time points of incubation showed that fast green FCF also exhibited disaggregating activity against the preformed/existing lysozyme fibrils. We believe that the results from this study suggest a potential therapeutic role of biocompatible molecules in treating or preventing protein aggregation diseases.

Polyhydroxycurcuminoids but not curcumin upregulate neprilysin and can be applied to the prevention of Alzheimer's disease.

Neprilysin (NEP) is the most important Aß-degrading enzyme. Its expression level decreases with age and inversely correlated with amyloid accumulation, suggesting its correlation with the late-onset of Alzheimer's disease. Recently, many reports showed that upregulating NEP level is a promising strategy in the prevention and therapy of Alzheimer's disease. Here, we used a sensitive fluorescence-based Aß digestion assay to screen 25 curcumin analogs for their ability to upregulate NEP activity. To our surprise, four compounds, dihydroxylated curcumin, monohydroxylated demethoxycurcumin, and mono- and di-hydroxylated bisdemethoxycurcumin, increased NEP activity, while curcumin did not. The ability of these polyhydroxycurcuminoids to upregulate NEP was further confirmed by mRNA and protein expression levels in the cell and mouse models. Finally, feeding monohydroxylated demethoxycurcumin (also named demethylcurcumin) or dihydroxylated bisdemethoxycurcumin (also named bisdemethylcurcumin) to APPswe/PS1dE9 double transgenic mice upregulated NEP levels in the brain and reduced Aß accumulation in the hippocampus and cortex. These polyhydroxycurcuminoids offer hope in the prevention of Alzheimer's disease.

Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy.

The conformation and dynamics of the unfolded state of ubiquitin doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated using single-molecule fluorescence spectroscopy. The line confocal fluorescence detection system combined with the rapid sample flow enabled the characterization of unfolded proteins at the improved structural and temporal resolutions compared to the conventional single-molecule methods. In the initial stage of the current investigation, however, the single-molecule Förster resonance energy transfer (sm-FRET) data of the labeled ubiquitin were flawed by artifacts caused by the adsorption of samples to the surfaces of the fused-silica flow chip and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl phosphorylcholine polymer to the flow chip surface was found to suppress the artifacts. The sm-FRET measurements based on the coated flow chip demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin at the native condition were narrowly distributed, which is comparable to the probability density function (PDF) expected from the shot noise, demonstrating the structural homogeneity of the native state. In contrast, the histogram of the sm-FRET efficiencies of the unfolded ubiquitin obtained at a time resolution of 100 µs was distributed significantly more broadly than the PDF expected from the shot noise, demonstrating the heterogeneity of the unfolded state conformation. The variety of the sm-FRET efficiencies of the unfolded state remained even after evaluating the moving average of traces with a window size of 1 ms, suggesting that conformational averaging of the heterogeneous conformations mostly occurs in the time domain slower than 1 ms. Local structural heterogeneity around the labeled fluorophores was inferred as the cause of the structural heterogeneity. The heterogeneity and slow dynamics revealed by the line confocal tracking of sm-FRET might be common properties of the unfolded proteins.