Molecular Chaperones and Mitochondrial Protein Folding

Precursor proteins destined for the mitochondrial matrix traverse inner and outer organelle membranes in an extended conformation. Translocation events are therefore integrally coupled to the processes of protein unfolding in the cytosol and protein refolding in the matrix. To successfully import proteins from the cytoplasm into mitochondria, cells have recruited a variety of molecular chaperone systems and folding catalysts. Within the organelles, mitochondrial Hsp70 (mt-Hsp70) is a major player in this process and exerts multiple functions. First, mt-Hsp70 binds together with cohort proteins to incoming polypeptide chains, thus conferring unidirectionality on the translocation process, and then assists in their refolding. A subset of imported proteins requires additional assistance by chaperonins of the Hsp60/Hsp10 family. Protein folding occurs within the cavity of these cylindrical complexes. A productive interaction of precursor proteins with molecular chaperones in the matrix is not only crucial for correct refolding and assembly, but also for processing of presequences, intramitochondrial sorting, and degradation of proteins. This review focuses on the role of mt-Hsp70 and Hsp60/Hsp10 in protein folding in the mitochondrial matrix and discusses recent findings on their molecular mechanism of action.     

The Dynamics of Genetic Draft in Rapidly Adapting Populations

The accumulation of beneficial mutations on competing genetic backgrounds in rapidly adapting populations has a striking impact on evolutionary dynamics. This effect, known as clonal interference, causes erratic fluctuations in the frequencies of observed mutations, randomizes the fixation times of successful mutations, and leaves distinct signatures on patterns of genetic variation. Here, we show how this form of “genetic draft” affects the forward-time dynamics of site frequencies in rapidly adapting asexual populations. We calculate the probability that mutations at individual sites shift in frequency over a characteristic timescale, extending Gillespie’s original model of draft to the case where many strongly selected beneficial mutations segregate simultaneously. We then derive the sojourn time of mutant alleles, the expected fixation time of successful mutants, and the site frequency spectrum of beneficial and neutral mutations. Finally, we show how this form of draft affects inferences in the McDonald–Kreitman test and how it relates to recent observations that some aspects of genetic diversity are described by the Bolthausen–Sznitman coalescent in the limit of very rapid adaptation.     

Evolutionary Aspects of Protein Structure and Folding

Four basic stages of evolution of protein structure are described, basing on recent work of the authors aimed specifically to reconstruct the earliest events in the protein evolution. According to this reconstruction, the initial stage of short peptides comprising, probably, only a few amino acid residues had been followed by formation of closed loops of 25–30 residues, which corresponds to the polymer-statistically optimal ring closure size for mixed polypeptide chains. The next stage involved fusion of relatively small linear genes and formation of protein structures consisting of several closed loops of a nearly standard size, with 4–6 loops (100–200 amino acid residues) in a typical protein fold. The last, modern stage began with combinatorial fusion of the presumably circular 300–600 bp DNA units and, accordingly, formation of multidomain proteins.     

Chaperones Rescue Luciferase Folding by Separating Its Domains

Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.     

Evolutionary Game Dynamics in Populations with Heterogenous Structures

Evolutionary graph theory is a well established framework for modelling the evolution of social behaviours in structured populations. An emerging consensus in this field is that graphs that exhibit heterogeneity in the number of connections between individuals are more conducive to the spread of cooperative behaviours. In this article we show that such a conclusion largely depends on the individual-level interactions that take place. In particular, averaging payoffs garnered through game interactions rather than accumulating the payoffs can altogether remove the cooperative advantage of heterogeneous graphs while such a difference does not affect the outcome on homogeneous structures. In addition, the rate at which game interactions occur can alter the evolutionary outcome. Less interactions allow heterogeneous graphs to support more cooperation than homogeneous graphs, while higher rates of interactions make homogeneous and heterogeneous graphs virtually indistinguishable in their ability to support cooperation. Most importantly, we show that common measures of evolutionary advantage used in homogeneous populations, such as a comparison of the fixation probability of a rare mutant to that of the resident type, are no longer valid in heterogeneous populations. Heterogeneity causes a bias in where mutations occur in the population which affects the mutant''s fixation probability. We derive the appropriate measures for heterogeneous populations that account for this bias.     

药物分子伴侣: 蛋白质折叠运输缺陷的新疗法

大量遗传性疾病的发生是由于基因突变引起蛋白质错误折叠而不能运输到作用位点,从而导致功能缺陷．近年来兴起的药物分子伴侣是恢复蛋白质折叠运输缺陷的新疗法,这类化合物一般为目的蛋白的底物类似物、受体配基或酶抑制剂等化学小分子,具细胞通透性,能在内质网中特异性识别并结合突变蛋白,校正并稳定其正确构象,协助其运输到正确位点,直接恢复突变蛋白功能,可治疗各种南蛋白质折叠运输缺陷导致的内分泌及代谢疾病．目前已报道的由药物分子伴侣恢复功能的突变蛋白主要为质膜蛋白及细胞器蛋白,如ATP结合盒转运蛋白、G-蛋白耦联受体及溶酶体酶等．大量的细胞及动物实验结果显示了药物分子伴侣的临床应用前景广阔,目前已有一例临床实验获得了成功．     

Coupled Protein Diffusion and Folding in the Cell

When a protein unfolds in the cell, its diffusion coefficient is affected by its increased hydrodynamic radius and by interactions of exposed hydrophobic residues with the cytoplasmic matrix, including chaperones. We characterize protein diffusion by photobleaching whole cells at a single point, and imaging the concentration change of fluorescent-labeled protein throughout the cell as a function of time. As a folded reference protein we use green fluorescent protein. The resulting region-dependent anomalous diffusion is well characterized by 2-D or 3-D diffusion equations coupled to a clustering algorithm that accounts for position-dependent diffusion. Then we study diffusion of a destabilized mutant of the enzyme phosphoglycerate kinase (PGK) and of its stable control inside the cell. Unlike the green fluorescent protein control''s diffusion coefficient, PGK''s diffusion coefficient is a non-monotonic function of temperature, signaling ‘sticking’ of the protein in the cytosol as it begins to unfold. The temperature-dependent increase and subsequent decrease of the PGK diffusion coefficient in the cytosol is greater than a simple size-scaling model suggests. Chaperone binding of the unfolding protein inside the cell is one plausible candidate for even slower diffusion of PGK, and we test the plausibility of this hypothesis experimentally, although we do not rule out other candidates.     

Induced Mutations in Yeast Cell Populations Adapting to an Unforeseen Challenge

The modern evolutionary synthesis assumes that mutations occur at random, independently of the environment in which they confer an advantage. However, there are indications that cells facing challenging conditions can adapt rapidly, utilizing processes beyond selection of pre-existing genetic variation. Here, we show that a strong regulatory challenge can induce mutations in many independent yeast cells, in the absence of general mutagenesis. Whole genome sequencing of cell lineages reveals a repertoire of independent mutations within a single lineage that arose only after the cells were exposed to the challenging environment, while other cells in the same lineage adapted without any mutation in their genomes. Thus, our experiments uncovered multiple alternative routes for heritable adaptation that were all induced in the same lineage during a short time period. Our results demonstrate the existence of adaptation mechanisms beyond random mutation, suggesting a tight connection between physiological and genetic processes.     

Continuous Probabilistic Analysis to Evolutionary Game Dynamics in Finite Populations

Evolutionary game dynamics of two strategies in finite population is studied by continuous probabilistic approach. Besides frequency dependent selection, mutation was also included in this study. The equilibrium probability density functions of abundance, expected time to extinction or fixation were derived and their numerical solutions are calculated as illustrations. Meanwhile, individual-based computer simulations are also done. A comparison reveals the consistency between theoretical analysis and simulations.     

Protein Folding Requires Crowd Control in a Simulated Cell

Macromolecular crowding has a profound effect upon biochemical processes in the cell. We have computationally studied the effect of crowding upon protein folding for 12 small domains in a simulated cell using a coarse-grained protein model, which is based upon Langevin dynamics, designed to unify the often disjoint goals of protein folding simulation and structure prediction. The model can make predictions of native conformation with accuracy comparable with that of the best current template-free models. It is fast enough to enable a more extensive analysis of crowding than previously attempted, studying several proteins at many crowding levels and further random repetitions designed to more closely approximate the ensemble of conformations. We found that when crowding approaches 40% excluded volume, the maximum level found in the cell, proteins fold to fewer native-like states. Notably, when crowding is increased beyond this level, there is a sudden failure of protein folding: proteins fix upon a structure more quickly and become trapped in extended conformations. These results suggest that the ability of small protein domains to fold without the help of chaperones may be an important factor in limiting the degree of macromolecular crowding in the cell. Here, we discuss the possible implications regarding the relationship between protein expression level, protein size, chaperone activity and aggregation.     

Rescue of Folding Defects in ABC Transporters Using Pharmacological Chaperones

The ATP-binding cassette (ABC) family of membrane transport proteins is the largest class of transporters in humans (48 members). The majority of ABC transporters function at the cell surface. Therefore, defective folding and trafficking of the protein to the cell surface can lead to serious health problems. The classic example is cystic fibrosis (CF). In most CF patients, there is a deletion of Phe508 in the CFTR protein (ΔF508 CFTR) that results in defective folding and intracellular retention of the protein (processing mutant). A potential treatment for most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect. The feasibility of such an approach was first demonstrated with the multidrug transporter P-glycoprotein (P-gp), an ABC transporter, and a sister protein of CFTR. It was found that P-gps with mutations at sites equivalent to those found in CFTR processing mutants were rescued when they were expressed in the presence of drug substrates or modulators of P-gp. These compounds acted as pharmacological chaperones and functioned by promoting interactions among the various domains in the protein during the folding process. Several groups have attempted to identify compounds that could rescue the folding defect in ΔF508 CFTR. The best compound identified through high-throughout screening is a quinazoline derivative (CFcor-325). Expression of ΔF508 CFTR as well as other CFTR processing mutants in the presence of 1 μM CFcor-325 promoted folding and trafficking of the mutant proteins to the cell surface in an active conformation. Therefore, CFcor-325 and other quinazoline derivates could be important therapeutic compounds for the treatment of CF.     

Multiplexed-Replica Exchange Molecular Dynamics Method for Protein Folding Simulation

Simulating protein folding thermodynamics starting purely from a protein sequence is a grand challenge of computational biology. Here, we present an algorithm to calculate a canonical distribution from molecular dynamics simulation of protein folding. This algorithm is based on the replica exchange method where the kinetic trapping problem is overcome by exchanging noninteracting replicas simulated at different temperatures. Our algorithm uses multiplexed-replicas with a number of independent molecular dynamics runs at each temperature. Exchanges of configurations between these multiplexed-replicas are also tried, rendering the algorithm applicable to large-scale distributed computing (i.e., highly heterogeneous parallel computers with processors having different computational power). We demonstrate the enhanced sampling of this algorithm by simulating the folding thermodynamics of a 23 amino acid miniprotein. We show that better convergence is achieved compared to constant temperature molecular dynamics simulation, with an efficient scaling to large number of computer processors. Indeed, this enhanced sampling results in (to our knowledge) the first example of a replica exchange algorithm that samples a folded structure starting from a completely unfolded state.     

Deleterious Passengers in Adapting Populations

Most new mutations are deleterious and are eventually eliminated by natural selection. But in an adapting population, the rapid amplification of beneficial mutations can hinder the removal of deleterious variants in nearby regions of the genome, altering the patterns of sequence evolution. Here, we analyze the interactions between beneficial “driver” mutations and linked deleterious “passengers” during the course of adaptation. We derive analytical expressions for the substitution rate of a deleterious mutation as a function of its fitness cost, as well as the reduction in the beneficial substitution rate due to the genetic load of the passengers. We find that the fate of each deleterious mutation varies dramatically with the rate and spectrum of beneficial mutations and the deleterious substitution rate depends nonmonotonically on the population size and the rate of adaptation. By quantifying this dependence, our results allow us to estimate which deleterious mutations will be likely to fix and how many of these mutations must arise before the progress of adaptation is significantly reduced.     

Extreme Selection Unifies Evolutionary Game Dynamics in Finite and Infinite Populations

We show that when selection is extreme—the fittest strategy always reproduces or is imitated—the unequivalence between the possible evolutionary game scenarios in finite and infinite populations resolves, in the sense that the three generic outcomes—dominance, coexistence, and mutual exclusion—emerge in well-mixed populations of any size. We consider the simplest setting of a 2-player-2-strategy symmetric game and the two most common microscopic definitions of strategy spreading—the frequency-dependent Moran process and the imitation process by pairwise comparison—both in the case allowing any intensity of selection. We show that of the seven different invasion and fixation scenarios that are generically possible in finite populations—fixation being more or less likely to occur and rapid compared to the neutral game—the three that are possible in large populations are the same three that occur for sufficiently strong selection: (1) invasion and fast fixation of one strategy; (2) mutual invasion and slow fixation of one strategy; (3) no invasion and no fixation. Moreover (and interestingly), in the limit of extreme selection 2 becomes mutual invasion and no fixation, a case not possible for finite intensity of selection that better corresponds to the deterministic case of coexistence. In the extreme selection limit, we also derive the large population deterministic limit of the two considered stochastic processes.     

Control of Cell Cycle and Cell Growth by Molecular Chaperones

Cells adapt their size to both intrinsic and extrinsic demands and, among them, those that stem from growth and proliferation rates are crucial for cell size homeostasis. Here we revisit mechanisms that regulate cell cycle and cell growth in budding yeast. Cyclin Cln3, the most upstream activator of Start, is retained at the endoplasmic reticulum in early G1 and released by specific chaperones in late G1 to initiate the cell cycle. On one hand, these chaperones are rate-limiting for release of Cln3 and cell cycle entry and, on the other hand, they are required for key biosynthetic processes. We propose a model whereby the competition for specialized chaperones between growth and cycle machineries could gauge biosynthetic rates and set a critical size threshold at Start.     

Molecular Dynamics Simulation of Protein Folding with Supersecondary Structure Constraints

We integrate molecular dynamics simulation methods with a newly developed supersecondary structure prediction method and compute the structure of a protein molecule, crambin. The computed structure is similar to the crystal structure with an rms error of 3.94 Å.