• Bulletin of the Korean Chemical Society
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• 제31권10호
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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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• 제38권3호
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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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• 제17권9호
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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.

• 미생물과산업
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• 제12권1호
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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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• 제32권2호
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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.

• 대한화학회지
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• 제63권6호
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• pp.427-434
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• 2019

단백질 폴딩 연구에 유용하도록 유비퀴틴 단백질의 페닐알라닌 45를 트립토판으로, 발린 26을 알라닌으로 변이시킨 HubWA 단백질을 모델로 삼아 소수성 상호작용이 단백질 폴딩 반응에 끼치는 영향을 탐구하였다. HubWA의 소수성 아미노산 중 14 개를 알라닌으로 치환한 변이 단백질을 제조하였고 이들 중 폴딩 연구에 적절한 4 개의 변이 단백질(V5A, I13A, V17A, I36A)을 얻어서 폴딩 반응의 진행 과정을 stopped-flow 장치로 측정하였다. 변이 단백질 V17A의 폴딩 반응은 HubWA와 마찬가지로 three-state 메커니즘을 따르며, V5A, I13A, I36A의 반응은 two-state 폴딩 메커니즘을 따르는 것으로 관찰되었다. 이는 HubWA 단백질의 폴딩 반응은 지엽적으로 구조적인 안정성을 지닌 부분이 존재하는 중간 단계가 먼저 형성된 다음 이들이 서로 퍼즐을 맞추는 것과 같은 방식으로 폴딩이 일어나는 collision-diffusion 메커니즘을 따르다가 소수성이 약한 아미노산으로 치환하였을 때 구조적인 안정성을 지닌 중간 단계가 관찰되지 않지만 폴딩 핵의 형성과 핵 주위로 native 구조가 형성되는 반응이 짝지어서 일어나는 nucleation-condensation 메커니즘으로 전환되는 것으로 해석되었다. 이러한 관찰은 단백질의 폴딩 경로는 지엽적인 구조의 안정성에 따라 서로 다른 메커니즘을 띨 수 있음을 시사한다.

• 대한화학회지
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• 제67권2호
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• pp.81-88
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• 2023

HubWA 단백질을 모델로 삼아 소수성 아미노산이 folding 반응에 끼치는 영향을 탐색하기 위하여 HubWA에 있는 I와 L을 V로 치환한 변이 단백질의 folding kinetics를 측정하였다. 변이 단백질의 folding kinetics는 HubWA 단백질과 마찬가지로 three-state on-pathway mechanism(U ⇌ I ⇌ N, U는 unfolded 상태, I는 중간단계, N은 native 상태를 의미한다)을 따르는 것으로 나타났다. Folding kinetics 분석을 통하여 three-state 반응의 elementary 반응과 전체 반응의 자유에너지인 ΔG^o_UI, ΔG^o_IN, ΔG^o_UN을 얻었고, 변이 단백질의 자유에너지와 HubWA 단백질의 자유에너지의 차(ΔΔG^o_UI = ΔG^o_UI(변이 단백질) - ΔG^o_UI(HubWA), ΔΔG^o_UN = ΔG^o_UN(변이 단백질) - ΔG^o_UN(HubWA))의 비인 ΔΔG^o_UI/ΔΔG^o_UN를 통하여 중간단계가 전체 folding 반응에 끼치는 영향을 각 소수성 잔기 별로 알아볼 수 있었다. HubWA의 입체구조에서 α-helix와 β-sheet가 상호작용하는 소수성 코어에 위치하는 아미노산인 I3, I13, L15, I30, L43, I61, L67을 V로 치환한 변이 단백질의 ΔΔG^o_UI/ΔΔG^o_UN 값이 ~0.5로 나타난 점은 이들 아미노산이 중간단계에서 native 상태보다는 느슨하지만 비교적 견고한 구조를 이루는 것으로 해석되었다. HubWA 입체구조에서 α-helix의 아미노말단에 위치하는 I23, 특정 이차구조가 없는 부위에 위치하는 I36, β-strand 5의 카복실말단에 위치하는 L69를 V로 치환한 변이 단백질의 ΔΔG^o_UI/ΔΔG^o_UN 값이 0.4 이하로 나타난 것은 이들 아미노산 잔기가 중간단계에서는 비교적 느슨한 구조를 이루다 중간단계에서 native 단계로 진행하는 folding 과정의 후반부에 HubWA의 입체구조에 견고하게 편입되는 것으로 해석되었다. HubWA의 입체구조에서 두 번째 β-strand의 카복실말단에 위치한 V17, 짧은 네 번째 β-strand의 카복실말단에 위치한 L50, 짧은 3₁₀-helix의 아미노말단에 위치한 L56이 중간단계에서 서로 상호작용을 하는 점은 이들 아미노산을 V로 치환한 변이 단백질의 ΔΔG^o_UI/ΔΔG^o_UN값이 0.8 이상으로 나타난 점을 통하여 알 수 있었다. L50과 L56은 짧은 β-strand와 3₁₀-helix를 제외하고 특별한 이차구조가 존재하지 않는 부위(46번째 아미노산 잔기부터 62번째 아미노산 잔기 까지)에 위치하는데, 이들 아미노산이 V17과 더불어 folding 반응의 초기에 견고하게 상호작용을 하는 것은 HubWA단백질이 folding 과정의 초기에 응집체를 형성하는 것을 막아주는 역할을하는 것으로 생각되었다.

• Bulletin of the Korean Chemical Society
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• 제35권6호
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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.

• 대한화학회지
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• 제58권6호
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• pp.560-568
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• 2014

단백질 폴딩 반응에서 정전기적 상호작용의 역할을 라이신 29를 알라닌으로 치환한 변이 유비퀴틴을 사용하여 탐색하였다. 유비퀴틴의 입체구조에서 라이신 29의 곁사슬은 글루탐산 16과 아스파르산 21의 곁사슬과 근접한 거리에 있어서 곁사슬끼리 정전기적 상호작용을 통하여서 삼차원 입체구조를 안정화시킬 것으로 예측되었다. 라이신을 알라닌으로 치환하여 정전기적 상호작용을 제거하였을 때 유비퀴틴의 native state의 구조적 안정성이 ~20% 감소한 점은 라이신 29에 의한 정전기적 상호작용이 단백질 삼차구조의 안정성에 상당히 기여하고 있다는 점을 시사하였다. 폴딩 반응의 진행 과정을 stopped-flow 장치로 측정한 folding kinetics 실험은 이전에 관찰된 것과 마찬가지로 unfolded state에서 native state로 진행하는 과정에 중간단계를 거치는 three-state on-pathway 메커니즘을 따르는 것으로 나타났다. 더욱이 라이신 29에 의한 정전기적 상호작용이 중간단계의 구조적 안정성에 기여하는 정도가 native state의 구조적 안정성에 기여하는 정도의 ~55%인 것으로 나타났다. 이는 유비퀴틴 폴딩의 중간단계의 구조도 라이신 29에 의한 정전기적 상호작용에 의하여 상당히 안정화 된다는 것을 의미하며 따라서 정전기적 상호작용이 단백질 삼차원 입체구조의 골격이 완성된 폴딩의 마지막 단계에 형성되어 단백질 native state의 안정성에만 기여하는 것이 아니라 중간단계가 형성되는 폴딩 반응의 초기에도 형성되어 폴딩 반응을 이끌어가는데도 기여한다는 것을 의미한다.

• BMB Reports
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• 제35권2호
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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.