• Bulletin of the Korean Chemical Society
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• v.31 no.10
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• pp.2877-2883
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• 2010

The folding kinetics of $WT^*$ ubiquitin variant with valine to alanine mutation at sequence position 26 (HubWA) was studied by stopped-flow fluorescence spectroscopy. While unfolding kinetics showed a single exponential phase, refolding reaction showed three exponential phases. The semi-logarithmic plot of urea concentration vs. rate constant for the first phase showed v-shape pattern while the second phase showed v-shape with roll-over effect at low urea concentration. The rate constant and the amplitude of the third phase were constant throughout the urea concentrations, suggesting that this phase represents parallel process due to the configurational isomerization. Interestingly, the first and second phases appeared to be coupled since the amplitude of the second phase increased at the expense of the amplitude of the first phase in increasing urea concentrations. This observation together with the roll-over effect in the second folding phase indicates the presence of intermediate state during the folding reaction of HubWA. Quantitative analysis of Hub-WA folding kinetics indicated that this intermediate state is on the folding pathway. Folding kinetics measurement of a mutant HubWA with hydrophobic core residue mutation, Val to Ala at residue position 17, suggested that the intermediate state has significant amount of native interactions, supporting the interpretation that the intermediate is on the folding pathway. It is considered that HubWA is a useful model protein to study the contribution of residues to protein folding process using folding kinetics measurements in conjunction with protein engineering.

• BMB Reports
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• v.38 no.3
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• pp.275-280
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• 2005

For most of proteins to be active, they need well-defined three-dimensional structures alone or in complex. Folding is a process through which newly synthesized proteins get to the native state. Protein folding inside cells is assisted by various chaperones and folding factors, and misfolded proteins are eliminated by the ubiquitin-proteasome degradation system to ensure high fidelity of protein expression. Under certain circumstances, misfolded proteins escape the degradation process, yielding to deposit of protein aggregates such as loop-sheet polymer and amyloid fibril. Diseases characterized by insoluble deposits of proteins have been recognized for long time and are grouped as conformational diseases. Study of protein folding mechanism is required for better understanding of the molecular pathway of such conformational diseases.

• Bulletin of the Korean Chemical Society
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• v.17 no.9
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• pp.808-813
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• 1996

To explore protein folding mechanism, we simulated a folding pathway in a simplified 3×3×3 cubic lattice. In the lattice folding Monte Carlo simulations, each of the 28 possible native packing pairs that exist in the native conformation was used as a conformational restraint. The native packing restraints in the lattice model could be considered as a disulfide linkage restraint in a real protein. The results suggest that proteins denatured with a small disulfide loop can, but not always, fold faster than proteins without any disulfide linkage and than proteins with a larger disulfide loop. The results also suggest that there is a rough correlation between loop size of the native packing restraint and folding time. That is, the order of native residue-residue packing interaction in protein folding is likely dependent on the residue-residue distance in primary sequence. The strength of monomer-monomer pairwise interaction is not important in the determination of the packing order in lattice folding. From the folding simulations of five strong folding lattice sequences, it was also found that the context encoded in the primary sequence, which we do not yet clearly understand, plays more crucial role in the determination of detailed folding kinetics. Our restrained lattice model approach would provide a useful strategy to the future protein folding experiments by suggesting a protein engineering for the fast or slow folding research.

• The Microorganisms and Industry
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• v.12 no.1
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• pp.9-14
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• 1986

We have adopted a genetic approach to identifying those residues and local sequences in a polypeptide chain which play an important role on the folding pathway. Our approach has been to isolate and characterize mutants which specifically alter the folding and subunit association pathway of a polypeptide chain, without altering the native protein. Such mutants distinguish residues involved in the kinetic control of conformation from residues involved in the stability and activity of the native protein. This approach is complementary to the efforts to characterize mutations which alter the stability of the mature protein(6,7,8). It is likely that many residues will have roles in both aspects of the functioning of the polypeptide chain. We thought it likely, however, that at least with large proteins, these aspects might be segregated in different local sequences.

• Biomolecules & Therapeutics
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• v.32 no.2
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• pp.183-191
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• 2024

The Unfolded Protein Response (UPR) serves as a critical cellular mechanism dedicated to maintaining protein homeostasis, primarily within the endoplasmic reticulum (ER). This pathway diligently responds to a variety of intracellular indicators of ER stress with the objective of reinstating balance by diminishing the accumulation of unfolded proteins, amplifying the ER's folding capacity, and eliminating slow-folding proteins. Prolonged ER stress and UPR irregularities have been linked to a range of neuropsychiatric disorders, including major depressive disorder, bipolar disorder, and schizophrenia. This review offers a comprehensive overview of the UPR pathway, delineating its activation mechanisms and its role in the pathophysiology of neuropsychiatric disorders. It highlights the intricate interplay within the UPR and its profound influence on brain function, synaptic perturbations, and neural developmental processes. Additionally, it explores evolving therapeutic strategies targeting the UPR within the context of these disorders, underscoring the necessity for precision and further research to effective treatments. The research findings presented in this work underscore the promising potential of UPR-focused therapeutic approaches to address the complex landscape of neuropsychiatric disorders, giving rise to optimism for improving outcomes for individuals facing these complex conditions.

• Journal of the Korean Chemical Society
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• v.63 no.6
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• pp.427-434
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• 2019

The role of hydrophobic residues on protein folding reaction was studied by folding kinetics measurements in conjunction with protein engineering. The HubWA, which was derived from human ubiquitin by mutating the residues at 45 (Phe to Trp) and 26 (Val to Ala), was used as a mutational background. Fourteen hydrophobic residues were mutated to alanine. Among fourteen variants generated, only four variant proteins (V5A, I13A, V17A, and I36A) were suitable for folding study. The folding kinetics of these variants was measured by stopped-flow fluorescence spectroscopy. The folding kinetics of HubWA and V17A was observed to follow a three-state on-pathway mechanism. On the other hand, folding kinetics of V5A, I13A, and I36A was observed to follow a two-state mechanism. Based on these observations, transition of protein folding reaction from collision-diffusion mechanism to nucleation-condensation mechanism was discussed.

• Journal of the Korean Chemical Society
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• v.67 no.2
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• pp.81-88
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• 2023

The contribution of hydrophobic residues to the protein folding reaction was studied by using HubWA variant proteins with I and L to V mutation. Folding kinetics of all V variant proteins was observed to be satisfied by a three-state on-pathway mechanism, U ⇌ I ⇌ N, where U, I, and N represent unfolded, intermediate, and native state, respectively. Three-state folding reaction was quantitatively analyzed and the free energy of folding of each elementary reactions and overall folding reaction, ΔG^o_UI, ΔG^o_IN, and ΔG^o_UN, were obtained. From the ratio of free energy difference between the variant protein and HubWA, ΔΔG^o_UI/ΔΔG^o_UN (ΔΔG^o_UI = ΔG^o_UI (variant protein) - ΔG^o_UI (HubWA) and ΔΔG^o_UN = ΔG^o_UN (variant protein) - ΔG^o_UN(HubWA)), the contribution of hydrophobic residues to HubWA folding was analyzed. The residues which are located in the hydrophobic core between α-helix and β-sheet, I3, I13, L15, I30, L43, I61 and L67, showed ΔΔG^o_UI/ΔΔG^o_UN value of ~0.5 when each of these residues was mutated to V, indicating that these residues form relatively solid hydrophobic core in the intermediate state. Residues located at the end of secondary structures and loop, I23, L69 and I36 showed ΔΔG^o_UI/ΔΔG^o_UN value below 0.4 when each of these residues was mutated to V, indicating that the region containing these residues are loosely formed in the intermediate state. V17A, L50V and L56V showed fairly high ΔΔG^o_UI/ΔΔG^o_UN value of ~0.8. Since L50 and L56 are located in the region containing long loop (residue 46 to 62), it is suggested that the high ΔΔG^o_UI/ΔΔG^o_UN value of these residues prevents the formation of aggregate at the early stage of folding reaction.

• Bulletin of the Korean Chemical Society
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• v.35 no.6
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• pp.1713-1719
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• 2014

1FSD is a 28-residue designed protein with a ${\beta}{\beta}{\alpha}$ motif. Since this protein displays most essential features of protein structures in such a small size, this model protein can be an outstanding system for evaluating the balance in the propensity of the secondary structures and the quality of all-atom protein force fields. Particularly, this protein would be difficult to fold to its correct native structure without establishing proper balances between the secondary structure elements in all-atom energy functions. In this work, a series of the recently optimized five amber protein force fields [$ff03^*$, $f99sb^*$-ildn, ff99sb-${\phi}^{\prime}$-ildn, ff99sb-nmr1-ildn, ff99sb-${\Phi}{\Psi}$(G24, CS)-ildn] were investigated for the simulations of 1FSD using a conventional molecular dynamics (MD) and a biased-exchange meta-dynamics (BEMD) methods. Among those tested force fields, we found that ff99sb-nmr1-ildn and ff99sb-${\Phi}{\Psi}$(G24, CS)-ildn are promising in that both force fields can locate the native state of 1FSD with a high accuracy (backbone rmsd ${\leq}1.7{\AA}$) in the global free energy minimum basin with a reasonable energetics conforming to a previous circular dichroism (CD) experiment. Furthermore, both force fields led to a common set of two distinct folding pathways with a heterogeneous nature of the transition state to the folding. We anticipate that these force fields are reasonably well balanced, thereby transferable to many other protein folds.

• Journal of the Korean Chemical Society
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• v.58 no.6
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• pp.560-568
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• 2014

The contribution of electrostatic interactions to protein folding reaction was studied by using mutant ubiquitin with lysine to alanine mutation at residue position 29. Based on the three dimensional structure of ubiquitin, lysine 29 is located close to negatively charged glutamate 16 and aspartate 21 and considered to stabilize the native state of ubiquitin by electrostatic interactions between these residues. The equilibrium unfolding experiment showed that the native stability was decreased by about ~20% upon mutation. This observation indicates lysine 29 indeed forms electrostatic interactions with nearby residues. Folding kinetics measurements using stopped-flow device and quantitative analysis of kinetics data indicate that ubiquitin folds from unfolded state to native state via intermediate state as observed previously. This intermediate state was observed to form immediately after the initiation of folding reaction. The folding intermediate was shown to be destabilized considerably upon lysine to alanine mutation. These observations indicate that electrostatic interactions can form early stage of protein folding and hence lead the folding reaction.

• BMB Reports
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• v.35 no.2
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• pp.143-154
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• 2002

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the $\alpha+\beta$ class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0-2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly $\beta$-sheet conformation and shows a strong binding to 8-anilino-1-napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.