A Zn-dependent structural transition of SOD1 modulates its ability to undergo phase separation.

The misfolding and mutation of Cu/Zn superoxide dismutase (SOD1) is commonly associated with amyotrophic lateral sclerosis (ALS). SOD1 can accumulate within stress granules (SGs), a type of membraneless organelle, which is believed to form via liquid-liquid phase separation (LLPS). Using wild-type, metal-deficient, and different ALS disease mutants of SOD1 and computer simulations, we report here that the absence of Zn leads to structural disorder within two loop regions of SOD1, triggering SOD1 LLPS and amyloid formation. The addition of exogenous Zn to either metal-free SOD1 or to the severe ALS mutation I113T leads to the stabilization of the loops and impairs SOD1 LLPS and aggregation. Moreover, partial Zn-mediated inhibition of LLPS was observed for another severe ALS mutant, G85R, which shows perturbed Zn-binding. By contrast, the ALS mutant G37R, which shows reduced Cu-binding, does not undergo LLPS. In addition, SOD1 condensates induced by Zn-depletion exhibit greater cellular toxicity than aggregates formed by prolonged incubation under aggregating conditions. Overall, our work establishes a role for Zn-dependent modulation of SOD1 conformation and LLPS properties that may contribute to amyloid formation.

Leishmania protein KMP-11 modulates cholesterol transport and membrane fluidity to facilitate host cell invasion.

The first step of successful infection by any intracellular pathogen relies on its ability to invade its host cell membrane. However, the detailed structural and molecular understanding underlying lipid membrane modification during pathogenic invasion remains unclear. In this study, we show that a specific Leishmania donovani (LD) protein, KMP-11, forms oligomers that bridge LD and host macrophage (MΦ) membranes. This KMP-11 induced interaction between LD and MΦ depends on the variations in cholesterol (CHOL) and ergosterol (ERG) contents in their respective membranes. These variations are crucial for the subsequent steps of invasion, including (a) the initial attachment, (b) CHOL transport from MΦ to LD, and (c) detachment of LD from the initial point of contact through a liquid ordered (Lo) to liquid disordered (Ld) membrane-phase transition. To validate the importance of KMP-11, we generate KMP-11 depleted LD, which failed to attach and invade host MΦ. Through tryptophan-scanning mutagenesis and synthesized peptides, we develop a generalized mathematical model, which demonstrates that the hydrophobic moment and the symmetry sequence code at the membrane interacting protein domain are key factors in facilitating the membrane phase transition and, consequently, the host cell infection process by Leishmania parasites.

Interplay of lipid head group and packing defects in driving amyloid-beta-mediated myelin-like model membrane deformation.

Accumulating evidence suggests that amyloid plaque-associated myelin lipid loss as a result of elevated amyloid burden might also contribute to Alzheimer's disease. The amyloid fibrils are closely associated with lipids under physiological conditions; however, the progression of membrane remodeling events leading to lipid-fibril assembly remains unknown. Here we first reconstitute the interaction of amyloid Beta 40 (Aß-40) with myelin-like model membrane and show that the binding of Aß-40 induces extensive tubulation. To look into the mechanism of membrane tubulation, we chose a set of membrane conditions varying in lipid packing density and net charge that allows us to dissect the contribution of lipid specificity of Aß-40 binding, aggregation kinetics, and subsequent changes in membrane parameters such as fluidity, diffusion, and compressibility modulus. We show that the binding of Aß-40 depends predominantly on the lipid packing defect densities and electrostatic interactions and results in rigidification of the myelin-like model membrane during the early phase of amyloid aggregation. Furthermore, elongation of Aß-40 into higher oligomeric and fibrillar species leads to eventual fluidization of the model membrane followed by extensive lipid membrane tubulation observed in the late phase. Taken together, our results capture mechanistic insights into snapshots of temporal dynamics of Aß-40-myelin-like model membrane interaction and demonstrate how short timescale, local phenomena of binding, and fibril-mediated load generation results in the consequent association of lipids with growing amyloid fibrils.

Conformational Switch of a Peptide Provides a Novel Strategy to Design Peptide Loaded Porous Organic Polymer for Pyroptosis Pathway Mediated Cancer Therapy.

While peptide-based drug development is extensively explored, this strategy has limitations due to rapid excretion from the body (or shorter half-life in the body) and vulnerability to protease-mediated degradation. To overcome these limitations, a novel strategy for the development of a peptide-based anticancer agent is introduced, utilizing the conformation switch property of a chameleon sequence stretch (PEP1) derived from a mycobacterium secretory protein, MPT63. The selected peptide is then loaded into a new porous organic polymer (PG-DFC-POP) synthesized using phloroglucinol and a cresol derivative via a condensation reaction to deliver the peptide selectively to cancer cells. Utilizing ensemble and single-molecule approaches, this peptide undergoes a transition from a disordered to an alpha-helical conformation, triggered by the acidic environment within cancer cells that is demonstrated. This adopted alpha-helical conformation resulted in the formation of proteolysis-resistant oligomers, which showed efficient membrane pore-forming activity selectively for negatively charged phospholipids accumulated in cancer cell membranes. The experimental results demonstrated that the peptide-loaded PG-DFC-POP-PEP1 exhibited significant cytotoxicity in cancer cells, leading to cell death through the Pyroptosis pathway, which is established by monitoring numerous associated events starting from lysosome membrane damage to GSDMD-induced cell membrane demolition. This novel conformational switch-based drug design strategy is believed to have great potential in endogenous environment-responsive cancer therapy and the development of future drug candidates to mitigate cancers.

Kinetoplastid Membrane Protein-11 Induces Pores in Anionic Phospholipid Membranes: Effect of Cholesterol.

Kinetoplastid membrane protein-11 (KMP-11), expressed in all stages of leishmanial life cycle, is considered a potential candidate for leishmaniasis vaccine. KMP-11 is found on the membrane surface of the parasite. Although the biological function of KMP-11 is unknown, we hypothesize from its sequence analysis that it may interact with the macrophage membrane and may influence the entry process of the parasite into the host cell. To validate this hypothesis, we have investigated the interaction of KMP-11 with unilamellar anionic phospholipid vesicles and explored its pore-forming activity. The decrease in negative ζ-potential of the vesicles and reduction in the fluorescence intensity of membrane-bound dye DiI C-18 suggest a strong association of KMP-11 with the membrane. The fluorescence leakage experiment as well as phase contrast microscopy shows direct evidence of KMP-11-induced pore formation in an anionic membrane. Incorporation of cholesterol into the membrane has been found to inhibit pore formation induced by KMP-11, suggesting an important role of cholesterol in leishmaniasis. Interestingly, vesicles containing only neutral phospholipid do not exhibit any tendency toward pore formation.

Network mapping of the conformational heterogeneity of SOD1 by deploying statistical cluster analysis of FTIR spectra.

A crucial contribution to the heterogeneity of the conformational landscape of a protein comes from the way an intermediate relates to another intermediate state in its journey from the unfolded to folded or misfolded form. Unfortunately, it is extremely hard to decode this relatedness in a quantifiable manner. Here, we developed an application of statistical cluster analyses to explore the conformational heterogeneity of a metalloenzyme, human cytosolic copper-zinc superoxide dismutase (SOD1), using the inputs from infrared spectroscopy. This study provides a quantifiable picture of how conformational information at one particular site (for example, the copper-binding pocket) is related to the information at the second site (for example, the zinc-binding pocket), and how this relatedness is transferred to the global conformational information of the protein. The distance outputs were used to quantitatively generate a network capturing the folding sub-stages of SOD1.

Understanding the evolutionary trend of intrinsically structural disorders in cancer relevant proteins as probed by Shannon entropy scoring and structure network analysis.

BACKGROUND: Malignant diseases have become a threat for health care system. A panoply of biological processes is involved as the cause of these diseases. In order to unveil the mechanistic details of these diseased states, we analyzed protein families relevant to these diseases. RESULTS: Our present study pivots around four apparently unrelated cancer types among which two are commonly occurring viz. Prostate Cancer, Breast Cancer and two relatively less frequent viz. Acute Lymphoblastic Leukemia and Lymphoma. Eight protein families were found to have implications for these cancer types. Our results strikingly reveal that some of the proteins with implications in the cancerous cellular states were showing the structural organization disparate from the signature of the family it constitutes. The sequences were further mapped onto respective structures and compared with the entropic profile. The structures reveal that entropic scores were able to reveal the inherent structural bias of these proteins with quantitative precision, otherwise unseen from other analysis. Subsequently, the betweenness centrality scoring of each residue from the structure network models was resorted to explore the changes in dependencies on residue owing to structural disorder. CONCLUSION: These observations help to obtain the mechanistic changes resulting from the structural orchestration of protein structures. Finally, the hydropathy indexes were obtained to validate the sequence space observations using Shannon entropy and in-turn establishing the compatibility.

Efficient Detection of Early Events of α-Synuclein Aggregation Using a Cysteine Specific Hybrid Scaffold.

In this study, we have designed and synthesized a new hybrid ligand (SCG) that can selectively detect cysteine in the free and protein-bound states within minutes at the subnanomolar level. Photoinduced electron transfer was responsible for the visible color change as well as a large increase in steady state fluorescence. This detection was validated by using multiple model protein systems with differing cysteine environments and spatial arrangements. SCG was able to monitor the early events of the folding/aggregation kinetics of α-synuclein, a protein involved in the pathology of Parkinson's disease. The early events consisted of conformational fluctuations between different forms of the protein and oligomer formation. SCG was found to be effective in detecting early isomers of α-syn in vitro and in live cell environments.

A novel tryptamine-appended rhodamine-based chemosensor for selective detection of Hg2+ present in aqueous medium and its biological applications.

A novel rhodamine-tryptamine conjugate-based fluorescent and chromogenic chemosensor (RTS) for detection of Hg2+ present in water was reported. After gradual addition of Hg2+ in aqueous methanol solution of RTS, a strong orange fluorescence and deep-pink coloration were observed. The probe showed high selectivity towards Hg2+ compared to other competitive metal ions. The 1:1 binding stoichiometry between RTS and Hg2+ was established by Job's plot analysis and mass spectroscopy. Initial studies showed that the synthesized probe RTS possessed fair non-toxicity and effectively passed through cell walls of model cell systems, viz., human neuroblastoma (SHSY5Y) cells and cervical cells (HeLa) to detect intercellular Hg2+ ions, signifying its utility in biological system. The limit of detection (LOD) was found to be 2.1 nM or 0.42 ppb by fluorescence titration. Additionally, the potential relevance of synthesized chemosensor for detecting Hg2+ ions in environmental water samples has been demonstrated. Graphical abstract ᅟ.

The Role of Intestinal Fatty Acid Binding Proteins in Protecting Cells from Fatty Acid Induced Impairment of Mitochondrial Dynamics and Apoptosis.

BACKGROUND/AIMS: The conformation, folding and lipid binding properties of the intestinal fatty acid binding proteins (IFABP) have been extensively investigated. In contrast, the functional aspects of these proteins are not understood and matter of debates. In this study, we aim to address the deleterious effects of FA overload on cellular components, particularly mitochondria; and how IFABP helps in combating this stress by restoring the mitochondrial dynamics. METHODS: In the present study the functional aspect of IFABP under conditions of lipid stress was studied by a string of extensive in-cell studies; flow cytometry by fluorescence-activated cell sorting (FACS), confocal imaging, western blotting and quantitative real time PCR. We deployed ectopic expression of IFABP in rescuing cells under the condition of lipid stress. Again in order to unveil the mechanistic insights of functional traits, we arrayed extensive computational approaches by means of studying centrality calculations along with protein-protein association and ligand induced cluster dissociation. While addressing its functional importance, we used FCS and in-silico computational analyses, to show the structural distribution and the underlying mechanism of IFABP's action. RESULTS: Ectopic expression of IFABP in HeLa cells has been found to rescue mitochondrial morphological dynamics and restore membrane potential, partially preventing apoptotic damage induced by the increased FAs. These findings have been further validated in the functionally relevant intestinal Caco-2 cells, where the native expression of IFABP protects mitochondrial morphology from abrogation induced by FA overload. However, this native level expression is insufficient to protect against apoptotic cell death, which is rescued, at least partially in cells overexpressing IFABP. In addition, shRNA mediated IFABP knockdown in Caco-2 cells compromises mitochondrial dynamics and switches on intrinsic apoptotic pathways under FA-induced metabolic stress. CONCLUSION: To summarize, the present study implicates functional significance of IFABP in controlling ligand-induced damage in mitochondrial dynamics and apoptosis.

Nanoparticle Induced Conformational Switch Between α-Helix and ß-Sheet Attenuates Immunogenic Response of MPT63.

Although significant efforts have been devoted to develop nanoparticle-based biopharmaceuticals, it is not understood how protein conformation and nanoparticle surface modulate each other in optimizing the activity and/or toxicity of the biological molecules. This is particularly important for a protein, which can adopt different conformational states separated by a relatively small energy barrier. In this paper, we have studied nanoparticle binding-induced conformational switch from ß-sheet to α-helix of MPT63, a small major secreted protein from Mycobacterium tuberculosis and a drug target against Tuberculosis. The binding of magnetite nanoparticles to MPT63 results in a ß-sheet to α-helix switch near the sequence stretch between the 19th and 30th amino acids. As a consequence, the immunogenic response of the protein becomes compromised, which could be restored by protein engineering. This study emphasizes that conformational stability toward NP surface binding may require optimization involving genetic engineering for development of a nanoparticle conjugated pharmaceutical.

Photoinduced Electron Transfer Switching Mechanism of a Naphthalimide Derivative with its Solvatochromic Behaviour: An Experimental and Theoretical Study with In Cell Investigations.

The sole existence of a t-bone-shaped naphthalimide derivative [2-(2-aminoethyl)-1H-benzo[de]isoquinoline-1,3(2H)dione] (NAP), which gives rise to a photoinduced electron transfer (PET) mechanism, has been established using a combination of experimental and theoretical studies. In parallel an in vitro-in cell PET mechanism has also been shown. To understand the photophysics of NAP, solvent studies have been carried out in different solvents. In addition, theoretical calculations have been conducted to explain the spectroscopic properties through optimized structures. A "turn off" PET mechanism has also been observed in the presence of specific metal ions, namely, Cr3+ , Fe3+ and Hg2+ among a series of metal ions. Theoretical studies reveal that NAP-Cr3+ , NAP-Fe3+ and NAP-Hg2+ have their HOMO energy states lying in between a HOMO-LUMO energy state of the t-bone-type NAP molecule. On the contrary, the HOMO state of the other metal ion-NAP conjugate (NAP-Mn+ ) does not lie in between the HOMO-LUMO energy gap of the t-bone-type NAP molecule. Coupled with in vitro studies, in cell investigations reveal an enhancement of fluorescence intensity of NAP upon cytosolic metal sensing. Furthermore, a very high cell viability of NAP treated cells as tested by MTT assay and a fast permeation of the said compound as revealed by flow cytometry suggest NAP to be a potential candidate in metal sensing and bioimaging applications.

Understanding the Effect of Single Cysteine Mutations on Gold Nanoclusters as Studied by Spectroscopy and Density Functional Theory Modeling.

Fluorescent metal nanoclusters have generated considerable excitement in nanobiotechnology, particularly in the applications of biolabeling, targeted delivery, and biological sensing. The present work is an experimental and computational study that aims to understand the effects of protein environment on the synthesis and electronic properties of gold nanoclusters. MPT63, a drug target of Mycobacterium tuberculosis, was used as the template protein to synthesize, for the first time, gold nanoclusters at a low micromolar concentration of the protein. Two single cysteine mutants of MPT63, namely, MPT63Gly20Cys (mutant I) and MPT63Gly40Cys (mutant II) were employed for this study. The experimental results show that cysteine residues positioned in two different regions of the protein induce varying electronic states of the nanoclusters depending on the surrounding amino acids. A mixture of five-atom and eight-atom clusters was generated for each mutant, and the former was found to be predominant in both cases. Computational studies, including density functional theory (DFT), frontier molecular orbital (FMO), and natural bond orbital (NBO) calculations, validated the experimental observations. The as-prepared protein-stabilized nanoclusters were found to have applications in the imaging of live cells.

The Non-native Helical Intermediate State May Accumulate at Low pH in the Folding and Aggregation Landscape of the Intestinal Fatty Acid Binding Protein.

There has been widespread interest in studying early intermediate states and their roles in protein folding. The interest in intermediate states has been further emphasized in the recent literature because of their implications for protein aggregation. Unfortunately, direct kinetic characterization of intermediates has been difficult because of the limited time resolutions offered by the kinetic techniques and the heterogeneity of the folding and aggregation landscape. Even in equilibrium experiments, the characterization of intermediate states could be difficult because (a) their populations in equilibrium could be low and/or (b) they lack any specific biochemical or biophysical signatures for their identification. In this paper, we have used fluorescence correlation spectroscopy to study the nature of a low-pH intermediate state of the intestinal fatty acid binding protein, a small protein with predominantly ß-sheet structure. Our results have shown that the pH 3 intermediate diffuses faster than the folded protein and has strong helix forming propensity. These behaviors support Lim's hypothesis according to which even an entirely ß-sheet protein would form helical bundles at the early stage. Using dynamic light scattering and thioflavin T binding measurements, we have observed that the pH 3 intermediate is prone to aggregation. We believe that early helix formation is the result of a local effect, which originates from the interaction of the neighboring amino acids around the hydrophobic core residues. This early intermediate reorganizes subsequently, and this structural reorganization is initiated by the destabilizing interactions induced by the distant residues, unfavorable entropic costs, and steric constraints of the hydrophobic side chains. Mutational analyses show further that the increase in the hydrophobicity in the hydrophobic core region increases the population of the α-helical intermediate, enhancing the aggregation propensity of the protein, while an identical change, distant from the hydrophobic core, does not show any effect. This study re-emphasizes an overlap between the folding and aggregation landscape of a protein, where the fine-tuning between the local and global effects may be important for the protein to fold efficiently or to aggregate.

Molecular Crowding Affects the Conformational Fluctuations, Peroxidase Activity, and Folding Landscape of Yeast Cytochrome c.

To understand how a protein folds and behaves inside living cells, the effects of synthetic crowding media on protein folding, function, stability, and association have been studied in detail. Because the effect of excluded volume is more prominent in an extended state than in the native protein, a majority of these studies have been conducted in the unfolded state of different model proteins. Here, we have used fluorescence correlation spectroscopy (FCS) and other biophysical methods to investigate the effect of crowding agents Ficoll70 and Dextran70 on the nativelike state of cytochrome c from yeast. Yeast cytochrome c (y-cytc) contains a substantial expanded state in its native folded condition, which is present in equilibrium with a compact conformer in aqueous buffer. We have found that the crowding medium affects the native state equilibrium between compact and expanded states, shifting its population toward the compact conformer. As a result, the peroxidase activity of y-cytc decreases. Urea-induced protein stability measurements show that the compaction destabilizes the protein due to charge repulsions between similar charged clusters. Interestingly, the time constant of conformational fluctuations between the compact and expanded conformers has been found to increase in the crowded milieu, suggesting a crucial role of the solution microviscosity.

Subtle Change in the Charge Distribution of Surface Residues May Affect the Secondary Functions of Cytochrome c.

Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c.

Studies of cytochrome c-551 unfolding using fluorescence correlation spectroscopy and other biophysical techniques.

In this paper, we have studied the equilibrium unfolding transitions of cytochrome c from Pseudomonas aeruginosa (cytc551), a small bacterial protein. Similar to eukaryotic cytochrome c, cytc551 folds sequentially, although significant differences exist in the order of folding units (foldons). There are two regions of cytc551 (N-terminal helix with residue number 3 to 10 and the loop 2 region containing residues 34 to 45), in which no foldon unit could be assigned. In addition, the helix containing the Cys-X-X-Cys-His motif, adjacent to the N-terminal helix (residue number 3 to 10), shows unexplained ultra-fast collapse. To obtain further insights, we have studied cytc551 site-directed mutants using fluorescence correlation spectroscopy (FCS) and molecular dynamics simulation. We have found out that cytc551 unfolds through the formation of a fluorescently dark intermediate state and the amplitude of the dark component depends on the position of labeling. We have utilized this position dependence to propose a shape change model during the unfolding of cytc551. The present results show that the N-terminal helix remains in a collapsed position even in the completely unfolded state and this helix may act as a rigid support to guide the folding of its adjacent helix. This rigid support may be responsible for the ultra-fast collapse of the adjacent helix region, which occurs during the initial events of folding. The present results also show that the C-terminal end of loop 2 traverses a large distance during unfolding compared to the N-terminal end, which justifies the observed flexibility of the loop 2 region.

Conversion of amyloid fibrils of cytochrome c to mature nanorods through a honeycomb morphology.

Amyloid species with various morphologies have been found for different proteins and disease systems. In this article, we aim to ask if these morphologies are unique to a particular protein or if they convert from one to another. Using a heme protein containing iron as the transition-metal activator of aggregation and a negatively charged surfactant, partial unfolding of the protein and its aggregation have been induced. In the pathway of aggregation, we have observed the formation of several morphological structures of a single protein, which were visualized directly using atomic force microscopy (AFM). These structures have been found to appear and disappear with time, and their formation could be monitored under normal buffer conditions and at room temperature without requiring any sophisticated chemical or biological methodologies. In addition, we have observed the formation of honeycomb-shaped morphology, which may serve as an intermediate. These amyloid-based nanostructures may have the potential to be explored in therapeutics delivery and other biomedical applications.

Attenuation of the early events of α-synuclein aggregation: a fluorescence correlation spectroscopy and laser scanning microscopy study in the presence of surface-coated Fe3O4 nanoparticles.

The aggregation of α-synuclein (A-syn) has been implicated in the pathogenesis of Parkinson's disease (PD). Although the early events of aggregation and not the matured amyloid fibrils are believed to be responsible for the toxicity, it has been difficult to probe the formation of early oligomers experimentally. We studied the effect of Fe3O4 nanoparticle (NP) in the early stage of aggregation of A-syn using fluorescence correlation spectroscopy (FCS) and laser scanning microscopy. The binding between the monomeric protein and NPs was also studied using FCS at single-molecule resolution. Our data showed that the addition of bare Fe3O4 NPs accelerated the rate of early aggregation, and it did not bind the monomeric A-syn. In contrast, L-lysine (Lys)-coated Fe3O4 NPs showed strong binding with the monomeric A-syn, inhibiting the early events of aggregation. Lys-coated Fe3O4 NPs showed significantly less cell toxicity compared with bare Fe3O4 NPs and can be explored as a possible strategy to develop therapeutic application against PD. To the best of our knowledge, this report is the first example of using a small molecule to attenuate the early (and arguably the most relevant in terms of PD pathogenesis) events of A-syn aggregation.

Studies of early events of folding of a predominately ß-sheet protein using fluorescence correlation spectroscopy and other biophysical methods.

The interplay between the early collapse of the unfolded state and the formation of the secondary structure has been the subject of extensive research in protein chemistry. In this study, we used the intestinal fatty acid binding protein (IFABP), a small model protein with predominately ß-sheet structure, to study the early events, including the early chain collapse and the formation of the secondary structure. We used a combination of fluorescence correlation spectroscopy and far-UV circular dichroism (CD) to understand how these early processes influence the late folding events like the stabilization of the secondary structure and aggregation. Acid-induced unfolded IFABP was found to collapse in the presence of low concentrations of added salt and aggregate at higher concentrations. Both the formation of the collapsed state and aggregation were conveniently probed by fluorescence correlation spectroscopy, a sensitive fluorescence technique with single-molecule resolution. In contrast, the formation of the secondary structure was monitored by far-UV CD. The results suggested that backbone hydrogen bond formation, not only the overall hydrophobicity of IFABP, may play crucial roles in the early collapse. Two mutant proteins positioned at a crucial nucleating site, namely, G80V and L64G, although being opposite in their overall hydrophobicity, collapsed relatively rapidly compared to the wild-type protein. The interconnection among the early collapse, the formation of the secondary structure, and aggregation was similar for these two mutants. Another mutant, G44V, which was identical in its overall hydrophobicity to G80V but situated in a region distant from the hydrophobic core, was found to be very different from G80V and L64G.