Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy

It is generally accepted that a globular protein in its native state adopts a single, well-defined conformation. However, there have been several reports that some proteins may exist in more than one distinct folded form in equilibrium. In the case of staphylococcal nuclease, evidence for multiple conformations has come from electrophoretic and NMR studies, although there has been some controversy as to whether these are actually interconvertible forms of the same molecular species. Recently, magnetization transfer (MT)-NMR has been developed as a means of studying the kinetics of conformational transitions in proteins. In the study reported here, this approach has been extended and used to demonstrate the presence of at least two native forms of nuclease in equilibrium and to study their interconversion with the unfolded state under the conditions of the thermal unfolding transition. The experiments reveal that two distinct native forms of the protein fold and unfold independently and that these can interconvert directly as well as via the unfolded state. The spectra of the different forms suggest that they are structurally similar but the MT experiments show that the kinetics of folding and unfolding are quite different. Characterization of this behaviour will, therefore, have important implications for our understanding of the relationship between structure and folding kinetics.     

从高分子构象与动力学到蛋白质折叠

针对作用在聚合物刷上的键拉力研究表明作用在接枝基面上的力随着聚合物刷接枝密度的增大反而减小,然而尾端单体上的拉伸张力并没有消失.高分子的构象和动力学转变决定了其物性和多种多样的应用,而生物大分子蛋白质作为由二十种不同属性的氨基酸构成的序列,更是具有由其序列所决定的特别的三维自然结构.本文就聚合物刷、聚合物纳米复合材料、聚合物网络等几种高分子体系的构象与动力学过程,及蛋白质构象和其折叠与去折叠的动力学过程做了介绍.特别是蛋白质的折叠与去折叠速率在单分子操纵实验中受到拉力的调控,通过测量这种拉力依赖的动力学过程、蛋白质的自由能曲面和折叠去折叠路径可以得到系统全面的研究.本文以肌肉蛋白titin的免疫球蛋白结构域I27为例对蛋白质折叠研究进行了阐述.     

The physical basis of how prion conformations determine strain phenotypes

A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathological consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiological effects. Here we present and experimentally validate an analytical model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion dilution, competition for a limited pool of soluble protein, and conformation-dependent differences in prion growth and division rates. Analysis of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiological impact for both infectious (prion) and non-infectious amyloids.     

Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA

The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.     

Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies

Atomic force microscopes and optical tweezers are widely used to probe the mechanical properties of individual molecules and molecular interactions, by exerting mechanical forces that induce transitions such as unfolding or dissociation. These transitions often occur under nonequilibrium conditions and are associated with hysteresis effects-features usually taken to preclude the extraction of equilibrium information from the experimental data. But fluctuation theorems allow us to relate the work along nonequilibrium trajectories to thermodynamic free-energy differences. They have been shown to be applicable to single-molecule force measurements and have already provided information on the folding free energy of a RNA hairpin. Here we show that the Crooks fluctuation theorem can be used to determine folding free energies for folding and unfolding processes occurring in weak as well as strong nonequilibrium regimes, thereby providing a test of its validity under such conditions. We use optical tweezers to measure repeatedly the mechanical work associated with the unfolding and refolding of a small RNA hairpin and an RNA three-helix junction. The resultant work distributions are then analysed according to the theorem and allow us to determine the difference in folding free energy between an RNA molecule and a mutant differing only by one base pair, and the thermodynamic stabilizing effect of magnesium ions on the RNA structure.     

Functional complexity and regulation through RNA dynamics

Changes to the conformation of coding and non-coding RNAs form the basis of elements of genetic regulation and provide an important source of complexity, which drives many of the fundamental processes of life. Although the structure of RNA is highly flexible, the underlying dynamics of RNA are robust and are limited to transitions between the few conformations that preserve favourable base-pairing and stacking interactions. The mechanisms by which cellular processes harness the intrinsic dynamic behaviour of RNA and use it within functionally productive pathways are complex. The versatile functions and ease by which it is integrated into a wide variety of genetic circuits and biochemical pathways suggests there is a general and fundamental role for RNA dynamics in cellular processes.     

Simultaneous determination of protein structure and dynamics

We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy--for obtaining experimental information at the atomic level about the structural and dynamical features of proteins--with the ability of molecular dynamics simulations to explore a wide range of protein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant advances in our ability to understand and utilize the structures of native proteins.     

Tetramerization of an RNA oligonucleotide containing a GGGG sequence.

Poly rG can form four-stranded helices. The Hoogsteen-paired quartets of G residues on which such structures depend are so stable that they will form in 5'-GMP solutions, provided that Na+ or K+ are present (see for example, refs 2-4). Telomeric DNA sequences, which are G-rich, adopt four-stranded antiparallel G-quartet conformations in vitro, and parallel tetramerization of G-rich sequences may be involved in meiosis. Here we show that RNAs containing short runs of Gs can also tetramerize. A 19-base oligonucleotide derived from the 5S RNA of Escherichia coli (strand III), 5'GCCGAUGGUAGUGUGGGGU3', forms a K(+)-stabilized tetrameric aggregate that depends on the G residues at its 3' end. This complex is so stable that it would be surprising if similar structures do not occur in nature.     

Misfolded proteins partition between two distinct quality control compartments

The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and prion disease, is thought to occur after failure of the cellular protein quality control mechanisms. Here we examine the formation of misfolded protein inclusions in the eukaryotic cytosol of yeast and mammalian cell culture models. We identify two intracellular compartments for the sequestration of misfolded cytosolic proteins. Partition of quality control substrates to either compartment seems to depend on their ubiquitination status and aggregation state. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Notably, disease-associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Enhancing ubiquitination of a prion protein suffices to promote its delivery to the juxtanuclear inclusion. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.     

Crystal structure of the transfer-RNA domain of transfer-messenger RNA in complex with SmpB

Accurate translation of genetic information into protein sequence depends on complete messenger RNA molecules. Truncated mRNAs cause synthesis of defective proteins, and arrest ribosomes at the end of their incomplete message. In bacteria, a hybrid RNA molecule that combines the functions of both transfer and messenger RNAs (called tmRNA) rescues stalled ribosomes, and targets aberrant, partially synthesized, proteins for proteolytic degradation. Here we report the 3.2-A-resolution structure of the tRNA-like domain of tmRNA (tmRNA(Delta)) in complex with small protein B (SmpB), a protein essential for biological functions of tmRNA. We find that the flexible RNA molecule adopts an open L-shaped conformation and SmpB binds to its elbow region, stabilizing the single-stranded D-loop in an extended conformation. The most striking feature of the structure of tmRNA(Delta) is a 90 degrees rotation of the TPsiC-arm around the helical axis. Owing to this unusual conformation, the SmpB-tmRNA(Delta) complex positioned into the A-site of the ribosome orients SmpB towards the small ribosomal subunit, and directs tmRNA towards the elongation-factor binding region of the ribosome. On the basis of this structure, we propose a model for the binding of tmRNA on the ribosome.     

Multiple conformations of proteins in native state

Examples of protein sequences that can adopt multiple native states are recently accumulated. Characterization of the protein multiple conformations will have important implications for our understanding of the relationship between structure and function, and their folding kinetics. In present review, the experimental evidence for the existence of multiple conformations in the native state of proteins, the molecular basis and the biological significance of multiple conformations of proteins are focused.     

Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy

Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, F?rster resonance energy transfer (FRET) has been measured on single, freely diffusing molecules. Here we use this method to determine properties of the free-energy surface for folding that have not been obtained from ensemble experiments. We show that single-molecule FRET measurements of a small cold-shock protein expose equilibrium collapse of the unfolded polypeptide and allow us to calculate limits on the polypeptide reconfiguration time. From these results, limits on the height of the free-energy barrier to folding are obtained that are consistent with a simple statistical mechanical model, but not with the barriers derived from simulations using molecular dynamics. Unlike the activation energy, the free-energy barrier includes the activation entropy and thus has been elusive to experimental determination for any kinetic process in solution.     

A Novel Structural Measure Separating Non-Coding RNAs from Genomic Backgrounds

RNA secondary structure has become the most exploitable feature for ab initio detection of non-coding RNA(nc RNA) genes from genome sequences. Previous work has used Minimum Free Energy(MFE) based methods developed to identify nc RNAs by measuring sequence fold stability and certainty. However, these methods yielded variable performances across different nc RNA species. Designing novel reliable structural measures will help to develop effective nc RNA gene finding tools. This paper introduces a new RNA structural measure based on a novel RNA secondary structure ensemble constrained by characteristics of native RNA tertiary structures. The new method makes it possible to achieve a performance leap from the previous structure-based methods. Test results on standard nc RNA datasets(benchmarks) demonstrate that this method can effectively separate most nc RNAs families from genome backgrounds.     

Mitochondrial class II introns encode proteins related to the reverse transcriptases of retroviruses

Organelle introns share several distinctive features that set them apart from their counterparts in nuclear-encoded pre-messenger RNAs (reviewed in ref. 1): their termini do not obey the GU...AG rule; the introns are 'structured' (members of the same family or 'class' can theoretically adopt very similar RNA secondary conformations and several of the postulated pairings have been confirmed by studies of splicing mutants and their revertants (see, for example, ref. 4); many introns from both classes contain long open reading frames. We report here that the proteins potentially encoded by four class II introns are related to several RNA-dependent polymerases of viral and transposable element origins.     

小蛋白天然结构与折叠速度关系的分子动力学模拟研究

用分子动力学模拟方法研究了小蛋白天然结构集合与其折叠速度的关系．根据蛋白质内存在接触的不同定义方式．利用分子动力学模拟方法得到了10个小蛋白的一系列构象集合,分析了其拓扑参数与折叠速度的关系,并与PDB单构象的情况进行了比较．用含主链重原子的方式定义接触,所计算的结果较好,天然结构集合所计算的拓扑参数与蛋白质折叠速度的关系可以更真实地反映实际情况．     

Energetic landscape of alpha-lytic protease optimizes longevity through kinetic stability.

During the evolution of proteins the pressure to optimize biological activity is moderated by a need for efficient folding. For most proteins, this is accomplished through spontaneous folding to a thermodynamically stable and active native state. However, in the extracellular bacterial alpha-lytic protease (alphaLP) these two processes have become decoupled. The native state of alphaLP is thermodynamically unstable, and when denatured, requires millennia (t1/2 approximately 1,800 years) to refold. Folding is made possible by an attached folding catalyst, the pro-region, which is degraded on completion of folding, leaving alphaLP trapped in its native state by a large kinetic unfolding barrier (t1/2 approximately 1.2 years). alphaLP faces two very different folding landscapes: one in the presence of the pro-region controlling folding, and one in its absence restricting unfolding. Here we demonstrate that this separation of folding and unfolding pathways has removed constraints placed on the folding of thermodynamically stable proteins, and allowed the evolution of a native state having markedly reduced dynamic fluctuations. This, in turn, has led to a significant extension of the functional lifetime of alphaLP by the optimal suppression of proteolytic sensitivity.     

Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration

Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.     

Protein folding and misfolding

The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends both on the intrinsic properties of the amino-acid sequence and on multiple contributing influences from the crowded cellular milieu. Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases.