Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar

Baxa, Michael C.,Yu, Wookyung,Adhikari, Aashish N.,Ge, Liang,Xia, Zhen,Zhou, Ruhong,Freed, Karl F.,Sosnick, Tobin R. National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.27

<P><B>Significance</B></P><P>An outstanding issue in protein science is identifying the relationship between sequence and folding, e.g., do sequences having similar structures have similar folding pathways? The homologs Proteins G & L have been cited as a primary example where sequence variations dramatically affect folding dynamics. However, our new results indicate that the homologs have similar folding behavior. At the highest point on the reaction surface, the pathways converge to similar ensembles. These findings are distinct from descriptions based on the widely used mutational ϕ analysis, partly due to nonnative behavior. Our study emphasizes that significant challenges remain both in characterizing and predicting transition state ensembles even for relatively simple proteins whose folding behavior is believed to be well understood.</P><P>Experimental and computational folding studies of Proteins L & G and NuG2 typically find that sequence differences determine which of the two hairpins is formed in the transition state ensemble (TSE). However, our recent work on Protein L finds that its TSE contains both hairpins, compelling a reassessment of the influence of sequence on the folding behavior of the other two homologs. We characterize the TSEs for Protein G and NuG2b, a triple mutant of NuG2, using ψ analysis, a method for identifying contacts in the TSE. All three homologs are found to share a common and near-native TSE topology with interactions between all four strands. However, the helical content varies in the TSE, being largely absent in Proteins G & L but partially present in NuG2b. The variability likely arises from competing propensities for the formation of nonnative β turns in the naturally occurring proteins, as observed in our <I>TerItFix</I> folding algorithm. All-atom folding simulations of NuG2b recapitulate the observed TSEs with four strands for 5 of 27 transition paths [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) <I>Science</I> 334(6055):517–520]. Our data support the view that homologous proteins have similar folding mechanisms, even when nonnative interactions are present in the transition state. These findings emphasize the ongoing challenge of accurately characterizing and predicting TSEs, even for relatively simple proteins.</P>

중간단계의 구조적 안정성을 통한 HubWA 단백질의 접힘 (folding) 반응 탐색

박순호 대한화학회 2023 대한화학회지 Vol.67 No.2

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 fold- ing reaction, ΔGoUI, ΔGoIN, and ΔGoUN, were obtained. From the ratio of free energy difference between the variant protein and HubWA, ΔΔGoUI/ΔΔGoUN (ΔΔGoUI = ΔGoUI (variant protein) − ΔGoUI (HubWA) and ΔΔGoUN = ΔGoUN (variant protein) − ΔGoUN (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 ΔΔGoUI/ΔΔGoUN 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 ΔΔGoUI/ΔΔGoUN 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 ΔΔGoUI/ΔΔGoUN 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 ΔΔGoUI/ΔΔGoUN value of these residues prevents the formation of aggregate at the early stage of folding reaction. HubWA 단백질을 모델로 삼아 소수성 아미노산이 folding 반응에 끼치는 영향을 탐색하기 위하여 HubWA에 있는 I와 L 을 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 ΔGoUI, ΔGoIN, ΔGoUN을 얻었고 , 변이 단백질의 자유에너지와 HubWA 단백질의 자유에너지의 차(ΔΔGoUI = ΔGoUI(변이 단백질 )–ΔGoUI(HubWA), ΔΔGoUN = ΔGoUN (변이 단백질 ) – ΔGoUN(HubWA))의 비인 ΔΔGoUI/ΔΔGoUN를 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 ΔΔGoUI/ΔΔGoUN 값이 ~0.5로 나타난 점은 이들 아미노산이 중간단계에서native 상태보다는 느슨하지만 비교적 견고한 구조를 이루는 것으로 해석되었다 . HubWA 입체구조에서 α-helix의 아미노말단에위치하는 I23, 특정 이차구조가 없는 부위에 위치하는 I36, β-strand 5의 카복실말단에 위치하는 L69를 V로 치환한 변이 단백질의 ΔΔGoUI/ΔΔGoUN 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 . HubWA의입체구조에서 두 번째 β-strand의 카복실말단에 위치한 V17, 짧은 네 번째 β-strand의 카복실말단에 위치한 L50, 짧은 310-helix의아미노말단에 위치한 L56이 중간단계에서 서로 상호작용을 하는 점은 이들 아미노산을 V로 치환한 변이 단백질의 ΔΔGoUI/ΔΔGoUN 값이 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.

Computational Three-Residue Fragment Assembly and Folding Optimization for Protein Structure Design

정민중 한국물리학회 2009 THE JOURNAL OF THE KOREAN PHYSICAL SOCIETY Vol.55 No.5

Computational modeling of the unique tertiary structure of a protein from its amino acid sequence alone is one of the important challenges in science and technology. The tertiary structure itself and the folding mechanism toward it are indispensable for understanding the function and the biological role of the protein. One of the computational methods often used for this modeling is the fragment assembly method because it shows good structural modeling performance in many cases. There are limitations, however, in the conventional fragment assembly methods. Arguments for uses of energy functions and global optimization to predict the structures are in progress, for example. In this study, a new modeling method to predict protein tertiary structures is proposed. The proposed system mainly consists of two methods. The first one is a fragment assembly, in which 3-residue fragments of representative protein chains are used to produce prototypes of a given sequence of amino acids. The second one is global optimization, which uses folding optimization to construct final protein structures. One of our computational models of the protein, which yielded a 5.15 °A root mean square deviation for its native tertiary structure, is provided with other experiments. Computational modeling of the unique tertiary structure of a protein from its amino acid sequence alone is one of the important challenges in science and technology. The tertiary structure itself and the folding mechanism toward it are indispensable for understanding the function and the biological role of the protein. One of the computational methods often used for this modeling is the fragment assembly method because it shows good structural modeling performance in many cases. There are limitations, however, in the conventional fragment assembly methods. Arguments for uses of energy functions and global optimization to predict the structures are in progress, for example. In this study, a new modeling method to predict protein tertiary structures is proposed. The proposed system mainly consists of two methods. The first one is a fragment assembly, in which 3-residue fragments of representative protein chains are used to produce prototypes of a given sequence of amino acids. The second one is global optimization, which uses folding optimization to construct final protein structures. One of our computational models of the protein, which yielded a 5.15 °A root mean square deviation for its native tertiary structure, is provided with other experiments.

Effective inter-residue contact definitions for accurate protein fold recognition

Yuan, Chao,Chen, Hao,Kihara, Daisuke BioMed Central 2012 BMC bioinformatics Vol.13 No.-

<P><B>Background</B></P><P>Effective encoding of residue contact information is crucial for protein structure prediction since it has a unique role to capture long-range residue interactions compared to other commonly used scoring terms. The residue contact information can be incorporated in structure prediction in several different ways: It can be incorporated as statistical potentials or it can be also used as constraints in ab initio structure prediction. To seek the most effective definition of residue contacts for template-based protein structure prediction, we evaluated 45 different contact definitions, varying bases of contacts and distance cutoffs, in terms of their ability to identify proteins of the same fold.</P><P><B>Results</B></P><P>We found that overall the residue contact pattern can distinguish protein folds best when contacts are defined for residue pairs whose Cβ atoms are at 7.0 Å or closer to each other. Lower fold recognition accuracy was observed when inaccurate threading alignments were used to identify common residue contacts between protein pairs. In the case of threading, alignment accuracy strongly influences the fraction of common contacts identified among proteins of the same fold, which eventually affects the fold recognition accuracy. The largest deterioration of the fold recognition was observed for β-class proteins when the threading methods were used because the average alignment accuracy was worst for this fold class. When results of fold recognition were examined for individual proteins, we found that the effective contact definition depends on the fold of the proteins. A larger distance cutoff is often advantageous for capturing spatial arrangement of the secondary structures which are not physically in contact. For capturing contacts between neighboring β strands, considering the distance between Cα atoms is better than the Cβ−based distance because the side-chain of interacting residues on β strands sometimes point to opposite directions.</P><P><B>Conclusion</B></P><P>Residue contacts defined by Cβ−Cβ distance of 7.0 Å work best overall among tested to identify proteins of the same fold. We also found that effective contact definitions differ from fold to fold, suggesting that using different residue contact definition specific for each template will lead to improvement of the performance of threading.</P>

Molecular chaperones maximize the native state yield on biological times by driving substrates out of equilibrium

Chakrabarti, Shaon,Hyeon, Changbong,Ye, Xiang,Lorimer, George H.,Thirumalai, D. National Academy of Sciences 2017 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.114 No.51

<P><B>Significance</B></P><P>Molecular chaperones have evolved to assist the folding of proteins and RNA, thus avoiding the deleterious consequences of misfolding. Thus, it is expected that increasing chaperone concentrations should enhance the yield of native states. While this has been observed in GroEL-mediated protein folding, experiments on <I>Tetrahymena</I> ribozyme folding assisted by CYT-19 surprisingly show the opposite trend. Here, we reconcile these divergent experimental observations by developing a unified theory of chaperone-assisted protein and RNA folding. We show that these ATP-fueled machines drive their substrates out of equilibrium, maximizing the nonequilibrium native yield in a given time rather than the absolute yield or folding rate. The theory predicts that in vivo the number of chaperones is regulated to optimize their functions.</P><P>Molecular chaperones facilitate the folding of proteins and RNA in vivo. Under physiological conditions, the in vitro folding of <I>Tetrahymena</I> ribozyme by the RNA chaperone CYT-19 behaves paradoxically; increasing the chaperone concentration reduces the yield of native ribozymes. In contrast, the protein chaperone GroEL works as expected; the yield of the native substrate increases with chaperone concentration. The discrepant chaperone-assisted ribozyme folding thus contradicts the expectation that it operates as an efficient annealing machine. To resolve this paradox, we propose a minimal stochastic model based on the Iterative Annealing Mechanism (IAM) that offers a unified description of chaperone-mediated folding of both proteins and RNA. Our theory provides a general relation that quantitatively predicts how the yield of native states depends on chaperone concentration. Although the absolute yield of native states decreases in the <I>Tetrahymena</I> ribozyme, the product of the folding rate and the steady-state native yield increases in both cases. By using energy from ATP hydrolysis, both CYT-19 and GroEL drive their substrate concentrations far out of equilibrium, thus maximizing the native yield in a short time. This also holds when the substrate concentration exceeds that of GroEL. Our findings satisfy the expectation that proteins and RNA be folded by chaperones on biologically relevant time scales, even if the final yield is lower than what equilibrium thermodynamics would dictate. The theory predicts that the quantity of chaperones in vivo has evolved to optimize native state production of the folded states of RNA and proteins in a given time.</P>

A Fusion Tag to Fold on: The S-Layer Protein SgsE Confers Improved Folding Kinetics to Translationally Fused Enhanced Green Fluorescent Protein

( Ristl Robin ),( Birgit Kainz ),( Gerhard Stadlmayr ),( Heinrich Sehuster ),( Dietmar Pum ),( Paul Messner ),( Christian Obinger ),( Christina Schaffer ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.9

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

Evidence of Interaction of Phage P22 Tailspike Protein with DnaJ During Translational Folding

( Lee Sang Chul ),( Myeong Hee Yu ) 한국미생물생명공학회 2004 Journal of microbiology and biotechnology Vol.14 No.1

Molecular Chaperones in Protein Quality Control

Lee, Suk-Yeong,Tsai, Francis T.F. Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.3

Proteins must fold into their correct three-dimensional conformation in order to attain their biological function. Conversely, protein aggregation and misfolding are primary contributors to many devastating human diseases, such as prion-mediated infections, Alzheimer's disease, type II diabetes and cystic fibrosis. While the native conformation of a polypeptide is encoded within its primary amino acid sequence and is sufficient for protein folding in vitro, the situation in vivo is more complex. Inside the cell, proteins are synthesized or folded continuously; a process that is greatly assisted by molecular chaperones. Molecular chaperones re a group of structurally diverse and mechanistically distinct proteins that either promote folding or prevent the aggregation of other proteins. With our increasing understanding of the proteome, it is becoming clear that the number of proteins that can be classified as molecular chaperones is increasing steadily. Many of these proteins have novel but essential cellular functions that differ from that of more 'conventional' chaperones, such as Hsp70 and the GroE system. This review focuses on the emerging role of molecular chaperones in protein quality control, i.e. the mechanism that rids the cell of misfolded or incompletely synthesized polypeptides that otherwise would interfere with normal cellular function.

Topological determinants of protein unfolding rates

Jung, Jaewoon,Lee, Jooyoung,Moon, Hie-Tae Wiley Subscription Services, Inc., A Wiley Company 2005 Proteins Vol.58 No.2

<P>For proteins that fold by two-state kinetics, the folding and unfolding processes are believed to be closely related to their native structures. In particular, folding and unfolding rates are influenced by the native structures of proteins. Thus, we focus on finding important topological quantities from a protein structure that determine its unfolding rate. After constructing graphs from protein native structures, we investigate the relationships between unfolding rates and various topological quantities of the graphs. First, we find that the correlation between the unfolding rate and the contact order is not as prominent as in the case of the folding rate and the contact order. Next, we investigate the correlation between the unfolding rate and the clustering coefficient of the graph of a protein native structure, and observe no correlation between them. Finally, we find that a newly introduced quantity, the impact of edge removal per residue, has a good overall correlation with protein unfolding rates. The impact of edge removal is defined as the ratio of the change of the average path length to the edge removal probability. From these facts, we conclude that the protein unfolding process is closely related to the protein native structure. Proteins 2005. © 2004 Wiley-Liss, Inc.</P>

Modulation of Intracellular Protein Activity at Level of Protein Folding by Beta-turn Engineering

Bharat Madan,이선구 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.3

Control of the intracellular protein activity is veryimportant in various biological studies and biotechnology. This has generally been achieved at the transcription andtranslation levels. Although control of the intracellularactivity at the protein folding level is conceptually possible,but there have been few studies. The present studyexamined this possibility by modulating the in vivo proteinfolding rate of green fluorescence protein (GFP) throughbeta-turn engineering. A type II’ two residue beta-turn inGFP was targeted to generate two sets of mutants. First, aswitch-off mutant was designed to stop the protein activitycompletely. The modulation mutants were then constructedto change the rates of GFP folding. The design of mutantswas based on the rationale that residues i+1 and i+2 of abeta-turn have defined residue preferences, and theirperturbation affects the rate of protein folding. The in vivofluorescence activity of the designed GFP variants wasswitched off and modulated as expected. The change in thein vivo folding patterns of the mutants was confirmed bySDS-PAGE and found to be similar to the intracellularfluorescence activities of the mutants. The in vitro refoldingkinetics performed with purified variants showed correlationswith the in vivo folding patterns. These results showed thatthe beta-turns in a protein can be a target for modulatingthe in vivo protein folding pattern and activity.