Construction of new ligand binding sites in proteins of known structure. II. Grafting of a buried transition metal binding site into Escherichia coli thioredoxin.

In an accompanying paper a computational procedure is described, which introduces new ligand-binding sites into proteins of known structure. Here we describe the experimental implementation of one of the designs, which is intended to introduce a copper-binding site into Escherichia coli thioredoxin. The new binding site can be introduced with a minimum of four amino acid changes. The binding site is buried so that structural rules for making mutations in the hydrophobic core of a protein, as well as for the introduction of new functions, are being tested in this experiment. The mutant protein is folded even in the absence of metals, and variants that retain the original activity of thioredoxin can be isolated. The protein has gained a metal-binding site specific for transition metals. The metal co-ordination chemistry at the binding site varies depending on the metal that is introduced into it. Mercury(II) is co-ordinated in the expected manner. Copper(II) binds in a way that was not anticipated in the original design. It appears to use two of the four residues intended to form the co-ordination sphere, and two other residues that were not part of the original set of mutations. It is therefore necessary not only to introduce new functional groups to form a new site, but also to consider and remove alternative modes of binding.     

Holding damaged DNA together.

The mammalian X-ray cross-complementing group 1 protein (XRCC1) is an important player in base excision repair of damaged DNA. Two new findings help to elucidate its role - biochemical data suggest that this multidomain protein interacts not only with three different enzymes, but also with the nicked DNA itself, and NMR data reveal the structure of the domain that interacts with both DNA polymerase beta and DNA.     

An application of capsid-specific artificial ankyrin repeat proteinproduced in E. coli for immunochromatographic assay as a surrogate for antibody

Immunochromatographic strip test is a unique type of rapid test that has been developed for use as part of a diagnostic kit for the rapid detection of antibodies and/or other proteins of interest. For the detection of target proteins, most of the commercial tests are assembled based on the conjugation of colloidal gold particles to monoclonal antibodies embedded within the conjugate pad of a strip test. In this study, we tested the novel concept of using an artificial non-antibody structure for generating a colloidal gold conjugate (CGC). We exploited the property of an ankyrin repeat protein that specifically binds to the HIV-1 capsid protein termed Ank^GAG1D4. This construct was applied as a model structure to create Ank1D4-CGC and used as a new type of visible detector system and termed it ankyrin-based immunochromatographic strip (ABIS) test. The ABIS test was shown to be highly sensitive with a lower limit of detection of the target protein at 0.1 μg/ml. Moreover, the ABIS test was not only highly sensitive but also shared a level of specificity within the same range of the commercial test kit. The results of the studies presented herein therefore demonstrate the novel application of an artificial non-immunoglobulin structure (ankyrin repeat protein) as the new line of a visible detector using a rapid diagnostic test with characteristics that have the potential to be superior to those that utilize antibody-based tests.     

Extracting Hydrogen-Bond Signature Patterns from Protein Structure Data

Classification of protein sequences and structures into families is a fundamental task in biology, and it is often used as a basis for designing experiments for gaining further knowledge. Some relationships between proteins are detected by the similarities in their sequences, and many more by the similarities in their structures. Despite this, there are a number of examples of functionally similar molecules without any recognisable sequence or structure similarities, and there are also a number of protein molecules that share common structural scaffolds but exhibit different functions. Newer methods of comparing molecules are required in order to detect similarities and dissimilarities in protein molecules. In this article, it is proposed that the precise 3-dimensional disposition of key residues in a protein molecule is what matters for its function, or what conveys the "meaning" for a biological system, but not what means it uses to achieve this. The concept of comparing two molecules through their intramolecular interaction networks is explored, since these networks dictate the disposition of amino acids in a protein structure. First, signature patterns, or fingerprints, of interaction networks in pre-classified protein structural families are computed using an approach to find structural equivalences and consensus hydrogen bonds. Five examples from different structural classes are illustrated. These patterns are then used to search the entire Protein Data Bank, an approach through which new, unexpected similarities have been found. The potential for finding relationships through this approach is highlighted. The use of hydrogen-bond fingerprints as a new metric for measuring similarities in protein structures is also described.     

Morphogenesis of the endoplasmic reticulum: beyond active membrane expansion

The endoplasmic reticulum (ER) of higher eukaryotic cells is a dynamic network of interconnected membrane tubules that pervades almost the entire cytoplasm. On the basis of the morphological changes induced by the disruption of the cytoskeleton or molecular motor proteins, the commonly accepted model has emerged that microtubules and conventional kinesin (kinesin-1) are essential determinants in establishing and maintaining the structure of the ER by active membrane expansion. Surprisingly, very similar ER phenotypes have now been observed when the cytoskeleton-linking ER membrane protein of 63 kDa (CLIMP-63) is mutated, revealing stable attachment of ER membranes to the microtubular cytoskeleton as a novel requirement for ER maintenance. Additional recent findings suggest that ER maintenance also requires ongoing homotypic membrane fusion, possibly controlled by the p97/p47/VICP135 protein complex. Work on other proteins proposed to regulate ER structure, including huntingtin, the EF-hand Ca(2+)-binding protein p22, the vesicle-associated membrane protein-associated protein B and kinectin isoforms further contribute to the new emerging concept that ER shape is not only determined by motor driven processes but by a variety of different mechanisms.     

Structural compliance: A new metric for protein flexibility

Proteins are the active players in performing essential molecular activities throughout biology, and their dynamics has been broadly demonstrated to relate to their mechanisms. The intrinsic fluctuations have often been used to represent their dynamics and then compared to the experimental B-factors. However, proteins do not move in a vacuum and their motions are modulated by solvent that can impose forces on the structure. In this paper, we introduce a new structural concept, which has been called the structural compliance, for the evaluation of the global and local deformability of the protein structure in response to intramolecular and solvent forces. Based on the application of pairwise pulling forces to a protein elastic network, this structural quantity has been computed and sometimes is even found to yield an improved correlation with the experimental B-factors, meaning that it may serve as a better metric for protein flexibility. The inverse of structural compliance, namely the structural stiffness, has also been defined, which shows a clear anticorrelation with the experimental data. Although the present applications are made to proteins, this approach can also be applied to other biomolecular structures such as RNA. This present study considers only elastic network models, but the approach could be applied further to conventional atomic molecular dynamics. Compliance is found to have a slightly better agreement with the experimental B-factors, perhaps reflecting its bias toward the effects of local perturbations, in contrast to mean square fluctuations. The code for calculating protein compliance and stiffness is freely accessible at https://jerniganlab.github.io/Software/PACKMAN/Tutorials/compliance .     

Routes are trees: the parsing perspective on protein folding

An important puzzle in structural biology is the question of how proteins are able to fold so quickly into their unique native structures. There is much evidence that protein folding is hierarchic. In that case, folding routes are not linear, but have a tree structure. Trees are commonly used to represent the grammatical structure of natural language sentences, and chart parsing algorithms efficiently search the space of all possible trees for a given input string. Here we show that one such method, the CKY algorithm, can be useful both for providing novel insight into the physical protein folding process, and for computational protein structure prediction. As proof of concept, we apply this algorithm to the HP lattice model of proteins. Our algorithm identifies all direct folding route trees to the native state and allows us to construct a simple model of the folding process. Despite its simplicity, our model provides an account for the fact that folding rates depend only on the topology of the native state but not on sequence composition.     

Structural stability of DNA in nonaqueous solvents

One of the defining physicochemical features of DNA in aqueous solution is its ability to maintain a double-helical structure and for this structure to undergo a cooperative, heat-induced denaturation (melting). Herein we show that a 21-mer synthetic DNA can form and maintain such a duplex structure not only in water but even in 99% glycerol; moreover, this double-helical structure reversibly and cooperatively melts in that solvent, with a T(m) value of some 30 degrees lower than in water. Two much larger, natural DNAs, from calf thymus and salmon testes, exhibit similar behavior in glycerol. All three DNAs can also sustain a double-helical structure in 99% ethylene glycol, although its thermostability (as reflected by the melting temperature) is some 20 degrees lower than in glycerol. In contrast, no duplex structure of any of the DNAs was detected in 99% formamide, methanol, or DMSO. This solvent trend resembles that previously observed in studies of protein structure and folding and underscores the importance of hydrophobic interactions in both protein and DNA structure and stability. Our findings suggest that water may not be unique as a suitable medium not only for protein structure but also for that of nucleic acids.     

Desperately seeking...something

Published online this week in Structure, Wisely et al. present a high-resolution X-ray crystallographic structure of the ligand binding domain of human hepatocyte nuclear factor 4 gamma (HNF4gamma). They find fatty acids filling the ligand binding pocket of this receptor long considered an orphan, but these "ligands" appear to be locked into the protein and not readily amenable to exchange. Not only does this present a new paradigm for nuclear receptors but it also provides new insights into their evolutionary origins.     

On the difference between mono-, holo-, and paraphyletic groups: a consistent distinction of process and pattern

About 50 years ago, the German entomologist Willi Hennig presented a new approach in biological systematics that he called a phylogenetic systematics. The main difference between his approach and traditional Linnean systematics was that he distinguished two new kinds of groups that he called mono- and paraphyletic groups, and whereof he considered only monophyletic groups to be natural groups. However, almost immediately after publication of his approach in English, some biological systematists commented that his monophyletic groups rather ought to be called holophyletic groups. The comment sparked a heated debate about the definition of the concept 'monophyletic groups', but the debate never reached consensus. In this paper, I claim that the controversy does not concern the definition of the concept monophyletic groups per se , but instead conceptualization of phylogenies (i.e. dichotomously branching processes) in a general sense. I discuss the relation between mono-, holo- and paraphyletic groups, and conclude that Hennig's conceptualization of phylogenies is both inconsistent and empirically wrong, whereas Linné's instead is consistent and correct.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 217–220.     

GP73 与消化系统疾病的研究进展

高尔基体是一个非常重要的细胞器,最近研究表明,它除了蛋白加工外,还能参与细胞分化、细胞间信号传导和细胞凋亡, 其功能障碍也许和疾病的发生、发展有着某种联系。随着蛋白组学技术的发展,使得筛选新的肿瘤标志物成为可能,其中高尔基 体蛋白73（GP73）被认为是最值得期待的血清标志物之一,尤其是早期肝癌的血清标志物。本文对近年来有关GP73 结构、表达分 布以与及消化系统疾病的研究进展进行综述。     

GP73与消化系统疾病的研究进展

高尔基体是一个非常重要的细胞器,最近研究表明,它除了蛋白加工外,还能参与细胞分化、细胞间信号传导和细胞凋亡,其功能障碍也许和疾病的发生、发展有着某种联系。随着蛋白组学技术的发展,使得筛选新的肿瘤标志物成为可能,其中高尔基体蛋白73（GP73）被认为是最值得期待的血清标志物之一,尤其是早期肝癌的血清标志物。本文对近年来有关GP73结构、表达分布以与及消化系统疾病的研究进展进行综述。     

Application of a new probabilistic model for recognizing complex patterns in glycans

MOTIVATION: The study of carbohydrate sugar chains, or glycans, has been one of slow progress mainly due to the difficulty in establishing standard methods for analyzing their structures and biosynthesis. Glycans are generally tree structures that are more complex than linear DNA or protein sequences, and evidence shows that patterns in glycans may be present that spread across siblings and into further regions that are not limited by the edges in the actual tree structure itself. Current models were not able to capture such patterns. RESULTS: We have applied a new probabilistic model, called probabilistic sibling-dependent tree Markov model (PSTMM), which is able to inherently capture such complex patterns of glycans. Not only is the ability to capture such patterns important in itself, but this also implies that PSTMM is capable of performing multiple tree structure alignments efficiently. We prove through experimentation on actual glycan data that this new model is extremely useful for gaining insight into the hidden, complex patterns of glycans, which are so crucial for the development and functioning of higher level organisms. Furthermore, we also show that this model can be additionally utilized as an innovative approach to multiple tree alignment, which has not been applied to glycan chains before. This extension on the usage of PSTMM may be a major step forward for not only the structural analysis of glycans, but it may consequently prove useful for discovering clues into their function.     

Correlated positions in protein evolution and engineering

Statistical analysis of a protein multiple sequence alignment can reveal groups of positions that undergo interdependent mutations throughout evolution. At these so-called correlated positions, only certain combinations of amino acids appear to be viable for maintaining proper folding, stability, catalytic activity or specificity. Therefore, it is often speculated that they could be interesting guides for semi-rational protein engineering purposes. Because they are a fingerprint from protein evolution, their analysis may provide valuable insight into a protein’s structure or function and furthermore, they may also be suitable target positions for mutagenesis. Unfortunately, little is currently known about the properties of these correlation networks and how they should be used in practice. This review summarises the recent findings, opportunities and pitfalls of the concept.     

The packing density in proteins: standard radii and volumes.

The sizes of atomic groups are a fundamental aspect of protein structure. They are usually expressed in terms of standard sets of radii for atomic groups and of volumes for both these groups and whole residues. Atomic groups, which subsume a heavy-atom and its covalently attached hydrogen atoms into one moiety, are used because the positions of hydrogen atoms in protein structures are generally not known. We have calculated new values for the radii of atomic groups and for the volumes of atomic groups. These values should prove useful in the analysis of protein packing, protein recognition and ligand design. Our radii for atomic groups were derived from intermolecular distance calculations on a large number (approximately 30,000) of crystal structures of small organic compounds that contain the same atomic groups to those found in proteins. Our radii show significant differences to previously reported values. We also use this new radii set to determine the packing efficiency in different regions of the protein interior. This analysis shows that, if the surface water molecules are included in the calculations, the overall packing efficiency throughout the protein interior is high and fairly uniform. However, if the water structure is removed, the packing efficiency in peripheral regions of the protein interior is underestimated, by approximately 3.5 %.     

Protein multiple alignment incorporating primary and secondary structure information.

Identifying common local segments, also called motifs, in multiple protein sequences plays an important role for establishing homology between proteins. Homology is easy to establish when sequences are similar (sharing an identity > 25%). However, for distant proteins, it is much more difficult to align motifs that are not similar in sequences but still share common structures or functions. This paper is a first attempt to align multiple protein sequences using both primary and secondary structure information. A new sequence model is proposed so that the model assigns high probabilities not only to motifs that contain conserved amino acids but also to motifs that present common secondary structures. The proposed method is tested in a structural alignment database BAliBASE. We show that information brought by the predicted secondary structures greatly improves motif identification. A website of this program is available at www.stat.purdue.edu/~junxie/2ndmodel/sov.html.     

Sarcolemmal Repair Is a Slow Process and Includes EHD2

Skeletal muscle is continually subjected to microinjuries that must be repaired to maintain structure and function. Fluorescent dye influx after laser injury of muscle fibers is a commonly used assay to study membrane repair. This approach reveals that initial resealing only takes a few seconds. However, by this method the process of membrane repair can only be studied in part and is therefore poorly understood. We investigated membrane repair by visualizing endogenous and GFP-tagged repair proteins after laser wounding. We demonstrate that membrane repair and remodeling after injury is not a quick event but requires more than 20 min. The endogenous repair protein dysferlin becomes visible at the injury site after 20 seconds but accumulates further for at least 30 min. Annexin A1 and F-actin are also enriched at the wounding area. We identified a new participant in the membrane repair process, the ATPase EHD2. We show, that EHD2, but not EHD1 or mutant EHD2, accumulates at the site of injury in human myotubes and at a peculiar structure that develops during membrane remodeling, the repair dome. In conclusion, we established an approach to visualize membrane repair that allows a new understanding of the spatial and temporal events involved.     

Potential for assessing quality of protein structure based on contact number prediction

We developed a novel knowledge-based residue environment potential for assessing the quality of protein structures in protein structure prediction. The potential uses the contact number of residues in a protein structure and the absolute contact number of residues predicted from its amino acid sequence using a new prediction method based on a support vector regression (SVR). The contact number of an amino acid residue in a protein structure is defined by the number of residues around a given residue. First, the contact number of each residue is predicted using SVR from an amino acid sequence of a target protein. Then, the potential of the protein structure is calculated from the probability distribution of the native contact numbers corresponding to the predicted ones. The performance of this potential is compared with other score functions using decoy structures to identify both native structure from other structures and near-native structures from nonnative structures. This potential improves not only the ability to identify native structures from other structures but also the ability to discriminate near-native structures from nonnative structures.     

Interaction of nucleotide excision repair factors RPA and XPA with DNA containing bulky photoreactive groups imitating damages

Interaction of nucleotide excision repair factors--replication protein A (RPA) and Xeroderma pigmentosum complementing group A protein (XPA)--with DNA structures containing nucleotides with bulky photoreactive groups imitating damaged nucleotides was investigated. Efficiency of photoaffinity modification of two proteins by photoreactive DNAs varied depending on DNA structure and type of photoreactive group. The secondary structure of DNA and, first of all, the presence of extended single-stranded parts plays a key role in recognition by RPA. However, it was shown that RPA efficiently interacts with DNA duplex containing a bulky substituent at the 5 -end of a nick. XPA was shown to prefer the nicked DNA; however, this protein was cross-linked with approximately equal efficiency by single-stranded and double-stranded DNA containing a bulky substituent inside the strand. XPA seems to be sensitive not only to the structure of DNA double helix, but also to a bulky group incorporated into DNA. The mechanism of damage recognition in the process of nucleotide excision repair is discussed.