The folding pathway of barnase: the rate-limiting transition state and a hidden intermediate under native conditions

The nature of the rate-limiting transition state at zero denaturant (TS(1)) and whether there are hidden intermediates are the two major unsolved problems in defining the folding pathway of barnase. In earlier studies, it was shown that TS(1) has small phi values throughout the structure of the protein, suggesting that the transition state has either a defined partially folded secondary structure with all side chains significantly exposed or numerous different partially unfolded structures with similar stability. To distinguish the two possibilities, we studied the effect of Gly mutations on the folding rate of barnase to investigate the secondary structure formation in the transition state. Two mutations in the same region of a beta-strand decreased the folding rate by 20- and 50-fold, respectively, suggesting that the secondary structures in this region are dominantly formed in the rate-limiting transition state. We also performed native-state hydrogen exchange experiments on barnase at pD 5.0 and 25 degrees C and identified a partially unfolded state. The structure of the intermediate was investigated using protein engineering and NMR. The results suggest that the intermediate has an omega loop unfolded. This intermediate is more folded than the rate-limiting transition state previously characterized at high denaturant concentrations (TS(2)). Therefore, it exists after TS(2) in folding. Consistent with this conclusion, the intermediate folds with the same rate and denaturant dependence as the wild-type protein, but unfolds faster with less dependence on the denaturant concentration. These and other results in the literature suggest that barnase folds through partially unfolded intermediates that exist after the rate-limiting step. Such folding behavior is similar to those of cytochrome c and Rd-apocyt b(562). Together, we suggest that other small apparently two-state proteins may also fold through hidden intermediates.     

Switching off the estrogen effect and approaches to its correction

Data are summarized on experimental means of induction of PSEE and its modification/prevention with the aim to achieve optimal ratio of estrogenic effects (as low as possible genotoxicity in combination with satisfactory and excessive hormonal action). Among studied agents were ethanol, tobacco smoke, irradiation, aging (as PSEE inductors) and carnosine, N-acetylcystein, vit. E and C, melatonin, swimming and antiestrogen ICI 182780 (as PSEE modificators).     

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).     

Insights into the structural nature of the transition state in the Kir channel gating pathway

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.     

Insights into the structural nature of the transition state in the Kir channel gating pathway

In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.     

Evolution of regulated phenotypic expression during a transition to multicellularity

Multicellular organisms coordinate growth and differentiation from single cell starting points with developmental programs. While the evolutionary origins of these programs are unknown, it is likely that they are closely tied to the evolution of regulated—not stochastic—phenotypic expression. To determine how such regulation might arise, we consider experimental populations of Pseudomonas fluorescens which evolved stochastic switching in the lab. This switching is directly coupled with environmental oscillations generated by the bacteria themselves. This unique example of niche construction provides reliable information that organisms may incorporate into regulation of phenotypes. We use mathematical models to investigate the success of two forms of regulation that rely on sensing either external or internal information. We find that both strategies can outcompete stochastic strategies for certain combinations of parameters. In particular, external sensing mechanisms are very effective over a large range of parameter space—including parameters that correspond to poor sensing of the extracellular signal and gradual responses. We show with evolutionary simulations that this robustness makes them more likely to evolve from initially stochastically switching populations rather than internal sensing mechanisms which require more tuning of parameters. These results demonstrate that, within this oscillating system, if regulatory mechanisms can evolve to incorporate environmental information then their selective advantage is sufficient for them to fix in the population.     

Modes of lactose uptake in the yeast species Kluyveromyces marxianus

Twelve lactose-assimilating strains of the yeast species Kluyveromyces marxianus and its varieties marxianus, lactis and bulgaricus were studied with respect to transport mechanisms for lactose, glucose and galactose, fermentation of these sugars and the occurrence of extracellular lactose hydrolysis. The strains fell into three groups. Group I (two strains): Fermentation of lactose, glucose and galactose, extracellular lactose hydrolysis, apparent facilitated diffusion of glucose and galactose; Group II (two strains): Lactose not fermented, glucose and galactose fermented and transported by an apparent proton symport, extracellular hydrolysis of lactose present (one strain) or questionable; Group III (eight strains): Lactose, glucose and galactose fermented, lactose transported by an apparent proton symport mechanism, extracellular hydrolysis of lactose and transport modes for glucose and galactose variable.     

Structured pathway across the transition state for peptide folding revealed by molecular dynamics simulations

Small globular proteins and peptides commonly exhibit two-state folding kinetics in which the rate limiting step of folding is the surmounting of a single free energy barrier at the transition state (TS) separating the folded and the unfolded states. An intriguing question is whether the polypeptide chain reaches, and leaves, the TS by completely random fluctuations, or whether there is a directed, stepwise process. Here, the folding TS of a 15-residue β-hairpin peptide, Peptide 1, is characterized using independent 2.5 μs-long unbiased atomistic molecular dynamics (MD) simulations (a total of 15 μs). The trajectories were started from fully unfolded structures. Multiple (spontaneous) folding events to the NMR-derived conformation are observed, allowing both structural and dynamical characterization of the folding TS. A common loop-like topology is observed in all the TS structures with native end-to-end and turn contacts, while the central segments of the strands are not in contact. Non-native sidechain contacts are present in the TS between the only tryptophan (W11) and the turn region (P7-G9). Prior to the TS the turn is found to be already locked by the W11 sidechain, while the ends are apart. Once the ends have also come into contact, the TS is reached. Finally, along the reactive folding paths the cooperative loss of the W11 non-native contacts and the formation of the central inter-strand native contacts lead to the peptide rapidly proceeding from the TS to the native state. The present results indicate a directed stepwise process to folding the peptide.     

The non-phagocytic route of collagen uptake: a distinct degradation pathway

The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.     

Electrostatic interactions in the denatured state and in the transition state for protein folding: effects of denatured state interactions on the analysis of transition state structure

The development of electrostatic interactions during the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) is investigated by pH-dependent rate equilibrium free energy relationships. We show that Asp8, among six acidic residues, is involved in non-native, electrostatic interactions with K12 in the transition state for folding as well as in the denatured state. The perturbed native state pK(a) of D8 (pK(a) = 3.0) appears to be maintained through non-native interactions in both the transition state and the denatured state. Mutational effects on the stability of the transition state for protein (un)folding are often analyzed in respect to change in ground states. Thus, the interpretation of transition state analysis critically depends on an understanding of mutational effects on both the native and denatured state. Increasing evidence for structurally biased denatured states under physiological conditions raises concerns about possible denatured state effects on folding studies. We show that the structural interpretation of transition state analysis can be altered dramatically by denatured state effects.     

Delivery of a lactose derivative of endomorphin 1 to the brain via the olfactory epithelial pathway

The rapid and direct delivery of a neuroactive endomorphin 1 derivative to the brain via nasal delivery is reported. A synthetic derivative of the native opioid peptide, endomorphin 1 bearing a lactose unit on the N-terminus of the peptide has been previously reported to exhibit antinoceceptive activity similar to morphine after both intravenous and oral administration. This compound has been administered nasally to rats and appeared in the olfactory bulb within 10 min of administration with negligible levels appearing in the circulating blood or in the rest of the brain. These results indicate that the peptide is absorbed into the brain via the olfactory epithelial pathway suggesting nasal delivery may be a viable alternative route of delivery in clinical applications.     

Mitochondrial permeability transition: a common pathway to necrosis and apoptosis

Opening of high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). The MPT is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca(2+) toxicity, and ischemia/reperfusion. CsA blocks opening of permeability transition pores and protects cell death after these stresses. In contrast to necrotic cell death which is a consequence of ATP depletion, ATP is required for the development of apoptosis. Reperfusion and the return of normal pH after ischemia initiate the MPT, but the balance between ATP depletion after the MPT and ATP generation by glycolysis determines whether the fate of cells will be apoptotic or necrotic death. Thus, the MPT is a common pathway leading to both necrotic and apoptotic cell death after ischemia/reperfusion.     

Mapping the folding pathway of an immunoglobulin domain: structural detail from Phi value analysis and movement of the transition state

BACKGROUND: Do proteins that have the same structure fold by the same pathway even when they are unrelated in sequence? To address this question, we are comparing the folding of a number of different immunoglobulin-like proteins. Here, we present a detailed protein engineering phi value analysis of the folding pathway of TI I27, an immunoglobulin domain from human cardiac titin. RESULTS: TI I27 folds rapidly via a kinetic intermediate that is destabilized by most mutations. The transition state for folding is remarkably native-like in terms of solvent accessibility. We use phi value analysis to map this transition state and show that it is highly structured; only a few residues close to the N-terminal region of the protein remain completely unfolded. Interestingly, most mutations cause the transition state to become less native-like. This anti-Hammond behavior can be used as a novel means of obtaining additional structural information about the transition state. CONCLUSIONS: The residues that are involved in nucleating the folding of TI I27 are structurally equivalent to the residues that form the folding nucleus in an evolutionary unrelated fibronectin type III protein. These residues form part of the common structural core of Ig-like domains. The data support the hypothesis that interactions essential for defining the structure of these beta sandwich proteins are also important in nucleation of folding.     

The role of respiration in tumor cell transition from the noncycling to the cycling state

Resting Yoshida AH130 hepatoma cells, harvested at the plateau of tumor development in vivo, were recruited into the cycling state following transfer to an in vitro system whereby these cells were incubated in the autologous ascites plasma diluted with buffered saline and enriched with glucose. In this system, cell recruitment into the phase of DNA synthesis (S phase) strictly depends on the activity of the respiratory chain and is abolished by anaerobiosis as well as by antimycin A, although the intracellular levels of ATP and the rate of protein synthesis are practically unaffected by these treatments. Furthermore, 2,4-dinitrophenol, at concentrations which uncouple the respiratory phosphorylation and hence enhance both glycolysis and oxygen consumption, does not hinder cell promotion into S phase. Thus, the absolute respiration dependence of cycling resumption by resting ascites cells does not seem to rely on respiratory ATP supply, but rather is linked to the electron flow through the respiratory chain.     

The slow S to M fluorescence rise in cyanobacteria is due to a state 2 to state 1 transition

In dark-adapted plants and algae, chlorophyll a fluorescence induction peaks within 1s after irradiation due to well documented photochemical and non-photochemical processes. Here we show that the much slower fluorescence rise in cyanobacteria (the so-called "S to M rise" in tens of seconds) is due to state 2 to state 1 transition. This has been demonstrated in particular for Synechocystis PCC6803, using its RpaC(-) mutant (locked in state 1) and its wild-type cells kept in hyperosmotic suspension (locked in state 2). In both cases, the inhibition of state changes correlates with the disappearance of the S to M fluorescence rise, confirming its assignment to the state 2 to state 1 transition. The general physiological relevance of the SM rise is supported by its occurrence in several cyanobacterial strains: Synechococcus (PCC 7942, WH 5701) and diazotrophic single cell cyanobacterium (Cyanothece sp. ATCC 51142). We also show here that the SM fluorescence rise, and also the state transition changes are less prominent in filamentous diazotrophic cyanobacterium Nostoc sp. (PCC 7120) and absent in phycobilisome-less cyanobacterium Prochlorococcus marinus PCC 9511. Surprisingly, it is also absent in the phycobiliprotein rod containing Acaryochloris marina (MBIC 11017). All these results show that the S to M fluorescence rise reflects state 2 to state 1 transition in cyanobacteria with phycobilisomes formed by rods and core parts. We show that the pronounced SM fluorescence rise may reflect a protective mechanism for excess energy dissipation in those cyanobacteria (e.g. in Synechococcus PCC 7942) that are less efficient in other protective mechanisms, such as blue light induced non-photochemical quenching. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

用差异显示PCR法筛选与血管外膜细胞表型转化相关的基因

为筛选血管外膜成纤维细胞（adventitial fibroblast,AF）与肌成纤维细胞（myofibroblast,MF）间表型转化有关的基因,实验建立了大鼠胸主动脉AF和MF两种细胞模型,用差异显示聚合酶链反应（DD－PCR）技术获得表达差异片段,对差异片段进行克隆和测序分析,并用定量PCR和Northern blot对差别显示结果进行验证。用反义核酸转染技术观察骨桥蛋白（osteopontin,OPN）对AF迁移的影响。结果表明,两种表型细胞存在明显的基因表达差异,其中一个在MF下调的差异片段与GenBank中NADH脱氢酶亚单位5（NADH dehydrogenase subunit 5,Nd5）基因高度同源。另一个在MF上调的差异片段与OPN基因同源。上述差异表达结果被定量PCR及Northern blot证实。此外还有4个表达序列标志（expressed sequence-tag,EST）在GenBank中未查到同源序列。反义OPN寡脱氧核甘酸可抑制AF的迁移活动。结果提示,AF转化为MF可能与ND5基因下调、OPN上调及其它未知基因的表达改变有关。应用反义技术适度抑制OPN表达在防治血管重塑中具有重要作用。