Evolutionary information for specifying a protein fold

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures. The artificial proteins show thermodynamic stabilities similar to natural WW domains, and structure determination of one artificial protein shows excellent agreement with the WW fold at atomic resolution. The relative simplicity of the information used for creating sequences suggests a marked reduction to the potential complexity of the protein-folding problem.     

Assessment of protein models with three-dimensional profiles.

As methods for determining protein three-dimensional (3D) structure develop, a continuing problem is how to verify that the final protein model is correct. The revision of several protein models to correct errors has prompted the development of new criteria for judging the validity of X-ray and NMR structures, as well as the formation of energetic and empirical methods to evaluate the correctness of protein models. The challenge is to distinguish between a mistraced or wrongly folded model, and one that is basically correct, but not adequately refined. We show that an effective test of the accuracy of a 3D protein model is a comparison of the model to its own amino-acid sequence, using a 3D profile, computed from the atomic coordinates of the structure 3D profiles of correct protein structures match their own sequences with high scores. In contrast, 3D profiles for protein models known to be wrong score poorly. An incorrectly modelled segment in an otherwise correct structure can be identified by examining the profile score in a moving-window scan. The accuracy of a protein model can be assessed by its 3D profile, regardless of whether the model has been derived by X-ray, NMR or computational procedures.     

A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron

Molecular self-assembly offers a means of spontaneously forming complex and well-defined structures from simple components. The specific bonding between DNA base pairs has been used in this way to create DNA-based nanostructures and to direct the assembly of material on the subnanometre to micrometre scale. In principle, large-scale clonal production of suitable DNA sequences and the directed evolution of sequence lineages towards optimized behaviour can be realized through exponential DNA amplification by polymerases. But known examples of three-dimensional geometric DNA objects are not amenable to cloning because they contain topologies that prevent copying by polymerases. Here we report the design and synthesis of a 1,669-nucleotide, single-stranded DNA molecule that is readily amplified by polymerases and that, in the presence of five 40-mer synthetic oligodeoxynucleotides, folds into an octahedron structure by a simple denaturation-renaturation procedure. We use cryo-electron microscopy to show that the DNA strands fold successfully, with 12 struts or edges joined at six four-way junctions to form hollow octahedra approximately 22 nanometres in diameter. Because the base-pair sequence of individual struts is not repeated in a given octahedron, each strut is uniquely addressable by the appropriate sequence-specific DNA binder.     

Natural-like function in artificial WW domains

Protein sequences evolve through random mutagenesis with selection for optimal fitness. Cooperative folding into a stable tertiary structure is one aspect of fitness, but evolutionary selection ultimately operates on function, not on structure. In the accompanying paper, we proposed a model for the evolutionary constraint on a small protein interaction module (the WW domain) through application of the SCA, a statistical analysis of multiple sequence alignments. Construction of artificial protein sequences directed only by the SCA showed that the information extracted by this analysis is sufficient to engineer the WW fold at atomic resolution. Here, we demonstrate that these artificial WW sequences function like their natural counterparts, showing class-specific recognition of proline-containing target peptides. Consistent with SCA predictions, a distributed network of residues mediates functional specificity in WW domains. The ability to recapitulate natural-like function in designed sequences shows that a relatively small quantity of sequence information is sufficient to specify the global energetics of amino acid interactions.     

Transfer of a beta-turn structure to a new protein context

Four-residue beta-turns and larger loop structures represent a significant fraction of globular protein surfaces and play an important role in determining the conformation and specificity of enzyme active sites and antibody-combining sites. Turns are an attractive starting point to develop protein design methods, as they involve a small number of consecutive residues, adopt a limited number of defined conformations and are minimally constrained by packing interactions with the remainder of the protein. The ability to substitute one beta-turn geometry for another will extend protein engineering beyond the redecoration of fixed backbone conformations to include local restructuring and the repositioning of surface side chains. To determine the feasibility and to examine the effect of such a structural modification on the fold and thermodynamic stability of a globular protein, we have substituted a five-residue turn sequence from concanavalin A for a type I' beta-turn in staphylococcal nuclease. The resulting hybrid protein is folded and has full nuclease enzymatic activity but reduced thermodynamic stability. The crystal structure of the hybrid protein reveals that the guest turn sequence retains the conformation of the parent concanavalin A structure when substituted in the nuclease host.     

Atom-by-atom analysis of global downhill protein folding

Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability and/or folding kinetics, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.     

四螺旋桨家族蛋白质序列——结构关系研究

蛋白质的三级结构唯一地由其氨基酸序列决定,这是广为人所接受的。然而,很多具有规则三级结构的蛋白质其氨基酸序列接近随机,这使得人们感到很困惑。本文将以四螺旋桨家族蛋白质为例,通过简化氨基酸残基,根据相似性方法把序列中的隐含对称性显示出来。结果表明氨基酸序列中的隐含对称性与三级结构的四重准对称性一致。     

改进的离散增量算法预测27类折叠子的结构类型

蛋白质二级结构预测是三级结构预测的一个非常重要的中间步骤,而折叠子识别和结构类型的准确预测则可以提高二级结构和三级结构预测的准确度.本文从蛋白质的一级序列出发,提出了一种改进的预测算法:以二肽组分、预测的二级结构信息、伪氨基酸组分和位置权重矩阵打分值等特征分别作为参数,输入离散增量算法的单分类器中,通过加权融合单分类器的计算结果,对27类折叠子的结构类型进行了预测,取得了较好的预测结果.     

Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism

DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.     

浙西北地区二叠系和三叠系的构造变形样式及其大地构造意义初步研究

浙西下三叠系统政棠组(T_1zh)发育有典型的鲍马序列,是一套深水相浊积岩建造;区域构造环境初步分析表明浙西北地区具被动大陆边缘沉积楔特征。下三叠系统政棠组(T_1zh)与二叠系协调变形,其构造变形样式总体上以向西北逆冲的冲褶席(duplex)~ 为特征,变形强度自南东向北西呈递减趋势。上三叠统乌灶组(T_3w)为含煤陆相磨拉石盆地建造,变形样式以宽缓褶皱和逆断层组合为主。该区大地构造相主要为前陆褶皱冲断带相,而土三叠统乌灶组(T_3w)为前陆磨拉石盆地相,二者对浙西北地区早中生代造山作用的研究具重要意义。     

质心法:受类别驱动的RNA二级结构预测方法

RNA二级结构预测问题是计算分子生物学中的一个重要问题.目前的RNA二级结构预测模型和算法都是把待测结构RNA的一级序列作为输入,仅根据输入的序列预测其二级结构.这样做丢失了待测RNA的类别信息,进而无法利用同类别RNA二级结构的保守性.在实际的生物学研究中,对于关心二级结构的RNA,其类别往往是已知的.本文提出一种新的RNA二级结构预测思路:结合类别信息、根据已知的近似形状细化RNA的二级结构.这种方法尤其适用于长度较短、保守性好的非编码RNA.该方法的一个关键问题是如何将一个序列按照近似的形状进行折叠.为此本文首次提出了茎区的"质心"和"质心距"的概念,并且给出了一个结合了Hopfield网络和类别信息的RNA二级结构预测算法.实验表明本文提出的方法在所测试ncRNA分子上效果好于目前的方法.     

Implications of thermodynamics of protein folding for evolution of primary sequences

Natural proteins exhibit essentially two-state thermodynamics, with one stable fold that dominates thermodynamically over a vast number of possible folds, a number that increases exponentially with the size of the protein. Here we address the question of whether this feature of proteins is a rare property selected by evolution or whether it is in fact true of a significant proportion of all possible protein sequences. Using statistical procedures developed to study spin glasses, we show that, given certain assumptions, the probability that a randomly synthesized protein chain will have a dominant fold (which is the global minimum of free energy) is a function of temperature, and that below a critical temperature the probability rapidly increases as the temperature decreases. Our results suggest that a significant proportion of all possible protein sequences could have a thermodynamically dominant fold.     

Solving the membrane protein folding problem

One of the great challenges for molecular biologists is to learn how a protein sequence defines its three-dimensional structure. For many years, the problem was even more difficult for membrane proteins because so little was known about what they looked like. The situation has improved markedly in recent years, and we now know over 90 unique structures. Our enhanced view of the structure universe, combined with an increasingly quantitative understanding of fold determination, engenders optimism that a solution to the folding problem for membrane proteins can be achieved.     

基于小波分析法的蛋白质结构研究

用小波的方法预测了一个已知结构蛋白质的二级结构，并把它同互连网蛋白质结构预测服务及其他结构预测软件得到的二级结构进行比较。结果表明：该方法可以较好地确定蛋白质的二级结构且不必进行同源蛋白质序列的联配。在预测未知结构的蛋白质序列方面，该方法与其他方法相比，预测结果并无显著差异，这说明小波分析法可以用于蛋白质结构研究，若与其他方法结合用于结构预测,将会起到更好的作用。     

Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy

A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-A resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (epsilon15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond epsilon15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.     

Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution.

The 30S ribosomal subunit binds messenger RNA and the anticodon stem-loop of transfer RNA during protein synthesis. A crystallographic analysis of the structure of the subunit from the bacterium Thermus thermophilus is presented. At a resolution of 5.5 A, the phosphate backbone of the ribosomal RNA is visible, as are the alpha-helices of the ribosomal proteins, enabling double-helical regions of RNA to be identified throughout the subunit, all seven of the small-subunit proteins of known crystal structure to be positioned in the electron density map, and the fold of the entire central domain of the small-subunit ribosomal RNA to be determined.     

Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis

The spliceosome is a dynamic macromolecular machine that assembles on pre-messenger RNA substrates and catalyses the excision of non-coding intervening sequences (introns). Four of the five major components of the spliceosome, U1, U2, U4 and U5 small nuclear ribonucleoproteins (snRNPs), contain seven Sm proteins (SmB/B', SmD1, SmD2, SmD3, SmE, SmF and SmG) in common. Following export of the U1, U2, U4 and U5 snRNAs to the cytoplasm, the seven Sm proteins, chaperoned by the survival of motor neurons (SMN) complex, assemble around a single-stranded, U-rich sequence called the Sm site in each small nuclear RNA (snRNA), to form the core domain of the respective snRNP particle. Core domain formation is a prerequisite for re-import into the nucleus, where these snRNPs mature via addition of their particle-specific proteins. Here we present a crystal structure of the U4 snRNP core domain at 3.6?? resolution, detailing how the Sm site heptad (AUUUUUG) binds inside the central hole of the heptameric ring of Sm proteins, interacting one-to-one with SmE-SmG-SmD3-SmB-SmD1-SmD2-SmF. An irregular backbone conformation of the Sm site sequence combined with the asymmetric structure of the heteromeric protein ring allows each base to interact in a distinct manner with four key residues at equivalent positions in the L3 and L5 loops of the Sm fold. A comparison of this structure with the U1 snRNP at 5.5?? resolution reveals snRNA-dependent structural changes outside the Sm fold, which may facilitate the binding of particle-specific proteins that are crucial to biogenesis of spliceosomal snRNPs.     

Folding-driven synthesis of oligomers.

The biological function of biomacromolecules such as DNA and enzymes depends on their ability to perform and control molecular association, catalysis, self-replication or other chemical processes. In the case of proteins in particular, the dependence of these functions on the three-dimensional protein conformation is long known and has inspired the development of synthetic oligomers and polymers with the capacity to fold in a controlled manner, but it remains challenging to design these so-called 'foldamers' so that they are capable of inducing or controlling chemical processes and interactions. Here we show that the stability gained from folding can be used to control the synthesis of oligomers from short chain segments reversibly ligated through an imine metathesis reaction. That is, folding shifts the ligation equilibrium in favour of conformationally ordered sequences, so that oligomers having the most stable solution structures form preferentially. Crystallization has previously been used to shift an equilibrium in order to indirectly influence the synthesis of small molecules, but the present approach to selectively prepare macromolecules with stable conformations directly connects folding and synthesis, emphasizing molecular function rather than structure in polymer synthesis.     

Three key residues form a critical contact network in a protein folding transition state

Determining how a protein folds is a central problem in structural biology. The rate of folding of many proteins is determined by the transition state, so that a knowledge of its structure is essential for understanding the protein folding reaction. Here we use mutation measurements--which determine the role of individual residues in stabilizing the transition state--as restraints in a Monte Carlo sampling procedure to determine the ensemble of structures that make up the transition state. We apply this approach to the experimental data for the 98-residue protein acylphosphatase, and obtain a transition-state ensemble with the native-state topology and an average root-mean-square deviation of 6 A from the native structure. Although about 20 residues with small positional fluctuations form the structural core of this transition state, the native-like contact network of only three of these residues is sufficient to determine the overall fold of the protein. This result reveals how a nucleation mechanism involving a small number of key residues can lead to folding of a polypeptide chain to its unique native-state structure.     

江苏靖江——苏州地区侏罗山式褶皱及滑脱断层的研究

研究区的褶皱是平行褶皱组成的隔档式褶皱;断裂以叠瓦状逆冲断层及撕裂断层的发育为特点。从几何学研究表明表层构造具有侏罗山式褶皱的特征,同时揭示其下必有滑脱断层的存在。椐运动学及动力学分析,该区构造是重力滑动的产物。