Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.     

Molecular Dynamics Simulations of Bromodomains Reveal Binding-Site Flexibility and Multiple Binding Modes of the Natural Ligand Acetyl-Lysine

Experimental protein structures provide spatial information at the atomic level. A further dimension, time, is supplemented by molecular dynamics. Since the pioneering work on the 58-residue inhibitor of bovine pancreatic trypsin in the group of Martin Karplus in the seventies, molecular dynamics simulations have shown that the intrinsic flexibility of proteins is essential for their function. Here, we review simulation studies of bromodomains. These protein modules are involved in the recognition of acetylated lysine side chains, a post-translational modification frequently observed in histone tails. The molecular dynamics simulations have unmasked: (i) the large plasticity of the loops lining the acetyl-lysine binding site (coupled to its self-occlusion), and (ii) multiple binding modes of acetyl-lysine. These simulation results suggest that recognition of histone tails by bromodomains is modulated by their intrinsic flexibility, and further corroborate the utility of molecular dynamics in understanding (macro)molecular recognition.     

Stereochemistry of the N-glycosylation sites in glycoproteins

The stereochemical features displayed by the N-glycosidic linkagein crystalline N-linked glycoproteins are analyzed. From thestatistical analysis of 44 different glycosylation sites belongingto 26 glycoproteins of the Brookhaven Protein Data Bank, a meanstandard geometry for the GlcNAc moiety, along with a rationalizationof its confornia-tional behavior, can be proposed. As for theglycopeptide linkage, the distribution of observed conformationshas been analyzed on the basis of molecular mechanics calculations.The rotamer distribution of the Asn side chains conforms tothat observed on non-glycosylated structures, and it agreeswith the pattern of flexible conformations gathered from NMRmeasurements. In characterizing the protein-glycan interactions,some hydrogen bonds occur. Stacking between the amphiphilicmoiety of the glycan and some surrounding aromatic, or at leasthydrophobic, amino acid residues is also found. When lookingat the secondary structure of the glycosylated peptide, only25% of the glycosylation sites correspond to situations whereAsn is located at the top of a ß-turn. Other typesof secondary structure exist which fulfil the spatial requirementof having the glycan exposed at the surface of the protein.These data can be compared with the most recent studies on thepeptide conformation which would be required for glycosylation.     

A Substantial Structural Conversion of the Native Monomer Leads to in-Register Parallel Amyloid Fibril Formation in Light-Chain Amyloidosis

Amyloid light-chain (AL) amyloidosis is a rare disease in which plasma-cell-produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid-associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid-state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show β-strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially ¹⁵N:¹³C-labeled samples, that the β-strands are stacked in-register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.     

Protein fold refinement: building models from idealized folds using motif constraints and multiple sequence data

A general solution to the problem of directly incorporatingdata from multiple sequence alignments into the constructionof molecular models was approached through the calculation ofan estimated pairwise distance based on conserved hydrophobidty.A scaling method was developed that allowed the required bulkgeometric properties of the estimated pair-wise distances (meanand mean squared) to mimic those expected in a globular protein.These properties were maintained independently of the composition,length, number or degree of conservation of the original sequences.Despite being a poor estimate for individual distances, thescaled distances were found to be compatible with the nativestructure and could be weighted highly. While the estimateddistances provided a general drive towards hydrophobk packing,more specific structures (including secondary structures andmotifs) were induced by regularization towards an ideal form.These constraints were used to refine an outline starting structure(derived only from secondary structure axes) towards a compactform that was sufficiently protein-like for side chains to beadded with almost no further adjustment of the a-carbon positions.This process allows rough folds based on abstract representationsof protein architecture to be rapidly converted to a form wherethey can be analysed by the growing number of methods designedto assess molecular models.     

Microstructure in solid polymers

Electron microscopy can only yield information about polymers in the solid state. Preparative methods have been developed which consisted of replica techniques; thin films stained with uranyl acetate, sodium tungstate or phosphotungstic acid; and extraction replicas. Results suggest an ordered sequence of structures from: (i) the stereochemically defined chain molecule which tends to form; (ii) a folded structure to minimise free energy; (iii) a structure which can be resolved in the electron microscope and is roughly spherical in shape-this structure is dependent on molecular weight up to a certain figure after which it is independent as is a constant for the material; and (iv) aggregates of structure (iii), either in the form of chains resembling a string of pearls or a pile of aggregated spheres.     

Current Understanding of the Structure,Stability and Dynamic Properties of Amyloid Fibrils

Amyloid fibrils are supramolecular protein assemblies represented by a cross-β structure and fibrous morphology, whose structural architecture has been previously investigated. While amyloid fibrils are basically a main-chain-dominated structure consisting of a backbone of hydrogen bonds, side-chain interactions also play an important role in determining their detailed structures and physicochemical properties. In amyloid fibrils comprising short peptide segments, a steric zipper where a pair of β-sheets with side chains interdigitate tightly is found as a fundamental motif. In amyloid fibrils comprising longer polypeptides, each polypeptide chain folds into a planar structure composed of several β-strands linked by turns or loops, and the steric zippers are formed locally to stabilize the structure. Multiple segments capable of forming steric zippers are contained within a single protein molecule in many cases, and polymorphism appears as a result of the diverse regions and counterparts of the steric zippers. Furthermore, the β-solenoid structure, where the polypeptide chain folds in a solenoid shape with side chains packed inside, is recognized as another important amyloid motif. While side-chain interactions are primarily achieved by non-polar residues in disease-related amyloid fibrils, the participation of hydrophilic and charged residues is prominent in functional amyloids, which often leads to spatiotemporally controlled fibrillation, high reversibility, and the formation of labile amyloids with kinked backbone topology. Achieving precise control of the side-chain interactions within amyloid structures will open up a new horizon for designing useful amyloid-based nanomaterials.     

Correlation between side chain mobility and conformation in protein structures

Thermal factors of protein atoms as determined by X-ray crystallographic techniques show a tendency to be larger in side chains with unfavourable local conformations rather than in those displaying conformational energy minima. It follows that side chain atoms are more mobile if they are in a non-rotameric configuration and that the stereochemistry of protein structures cannot be fully assessed or simulated without consideration of thermal factors that monitor flexibility in various regions of the protein. The observations should also prove useful in protein folding and design.      

聚羧酸类减水剂的分子设计与结构性能关系

根据高性能减水剂分子设计原理,通过甲基丙烯酸、甲基丙烯磺酸钠、甲氧基聚乙二醇丙烯酸酯等单体的自由基溶液聚合制备了分子链中含有聚氧乙烯链和阴离子基团的聚羧酸类减水剂。结果表明,接枝链的密度和侧链的长度影响减水剂的性能,通过选择适宜长度的侧链及调节共聚单体的比例和聚合物分子量可以增大减水剂的分散性和分散保持性。另外还通过红外光谱对产物进行了表征和分析,结果表明已得到预期结构的聚羧酸类减水剂。     

Grow to Fit Molecular Dynamics (G2FMD): an ab initio method for protein side-chain assignment and refinement

The rough energy landscapes and tight packing of protein interiors are two of the critical factors that have prevented the wide application of physics-based models in protein side-chain assignment and protein structure prediction in general. Complementing the rotamer-based methods, we propose an ab initio method that utilizes molecular mechanics simulations for protein side-chain assignment and refinement. By reducing the side-chain size, a smooth energy landscape was obtained owing to the increased distances between the side chains. The side chains then gradually grow back during molecular dynamics simulations while adjusting to their surrounding driven by the interaction energies. The method overcomes the barriers due to tight packing that limit conformational sampling of physics-based models. A key feature of this approach is that the resulting structures are free from steric collisions and allow the application of all-atom models in the subsequent refinement. Tests on a small set of proteins showed nearly 100% accuracy on both chi1 and chi2 of buried residues and 94% of them were within 20 degrees from the native conformation, 79% were within 10 degrees and 42% were within 5 degrees . However, the accuracy decreased when exposed side chains were involved. Further improvement and application of the method and the possible reasons that affect the accuracy on the exposed side chains are discussed.     

Supramolecular approach for synthesis and functionalization of cyclic macromolecules

This report highlights our recent findings concerning the synthesis of cyclic polymers based on supramolecular chemistry as well as the stereochemical recognition of helices by a cyclic macromolecule consisting of helical peptides and porphyrins. The first part will focus on an electrostatic self-assembly and covalent fixation strategy for the efficient synthesis of cyclic polymers. It has been shown that a unimeric polymer assembly is formed exclusively from the linear polymer precursor having cyclic ammonium salt end groups carrying dicarboxylate counter ions. An effective synthesis of cyclic polymers has been achieved by the subsequent covalent transformation of the ammonium salt groups. The process has been extended to the synthesis of cyclic macromonomers, which produced a unique polymer network having both covalent and mechanical linkages. The second part will focus on the stereochemical recognition of helices by a cyclic host. α-Aminoisobutyric acid (Aib) peptide-based cyclic hosts having metalloporphyrin units have been synthesized for guest binding and chiroptical sensing. By using these cavities, biologically important “peptide bundling” has been realized through complexation of helical peptide guests, where the three helical chains, two of which are from the host and one from the guest, are harmonized stereochemically in a confined cavity, leading to intense chiroptical outputs in the absorption bands of the metalloporphyrin units. The selective peptide bundling events based on helical senses of the host/guest molecules has also been achieved with a chiral conformational matching.     

In search of the ideal protein sequence

The inverse of a folding problem is to find the ideal sequencethat folds into a particular protein structure. This problemhas been addressed using the topology fingerprintbased threadingalgorithm, capable of calculating a score (energy) of an arbitrarysequence-structure pair. At first, the search is conducted byunconstrained minimization of the energy in sequence space.It is shown that using energy as the only design criterion leadsto spurious solutions with incorrect amino acid composition.The problem lies in the general features of the protein energysurface as a function of both structure and sequence. The proposedsolution is to design the sequence by maximizing the differencebetween its energy in the desired structure and in other knownprotein structures. Depending on the size of the database ofstructures ‘to avoid’, sequences bearing significantsimilarity to the native sequence of the target protein areobtained using this procedure.     

Recent Progress in the Synthesis of Homogeneous Erythropoietin (EPO) Glycoforms

Erythropoietin (EPO) has been regarded as a therapeutic glycoprotein for the clinical treatment of anaemia since its approval by the Food and Drug Administration (FDA) in 1989. Commercial production of the 165-residue glycoprotein is by recombinant protein expression using mammalian cell lines that renders a complex mixture of glycoforms that have an identical amino acid sequence but variations in the structures of the pendant glycans. This heterogeneous nature of human recombinant EPO restricts structural and bioactivity studies in medicinal chemistry. Consequently, chemical synthesis provides an elegant approach for the preparation of complex homogeneous glycoproteins from a readily accessible pool of amino acids and sugars. In addition, the combination of chemical and biosynthesis enables robust and large-scale production of homogeneous EPO. The scope of this minireview is to summarise the recent advances in the chemical and semisyntheses of homogeneous EPO glycoforms, highlighting the versatile approaches to the preparation and structural manipulations of the carbohydrate chains incorporated into synthetic EPO glycoproteins.     

Slipknot or Crystallographic Error: A Computational Analysis of the Plasmodium falciparum DHFR Structural Folds

The presence of protein structures with atypical folds in the Protein Data Bank (PDB) is rare and may result from naturally occurring knots or crystallographic errors. Proper characterisation of such folds is imperative to understanding the basis of naturally existing knots and correcting crystallographic errors. If left uncorrected, such errors can frustrate downstream experiments that depend on the structures containing them. An atypical fold has been identified in P. falciparum dihydrofolate reductase (PfDHFR) between residues 20–51 (loop 1) and residues 191–205 (loop 2). This enzyme is key to drug discovery efforts in the parasite, necessitating a thorough characterisation of these folds. Using multiple sequence alignments (MSA), a unique insert was identified in loop 1 that exacerbates the appearance of the atypical fold-giving it a slipknot-like topology. However, PfDHFR has not been deposited in the knotted proteins database, and processing its structure failed to identify any knots within its folds. The application of protein homology modelling and molecular dynamics simulations on the DHFR domain of P. falciparum and those of two other organisms (E. coli and M. tuberculosis) that were used as molecular replacement templates in solving the PfDHFR structure revealed plausible unentangled or open conformations of these loops. These results will serve as guides for crystallographic experiments to provide further insights into the atypical folds identified.     

Variations of the 2-His-1-carboxylate theme in mononuclear non-heme FeII oxygenases

A facial triad of two histidine side chains and one aspartate or glutamate side chain forms the canonical metal-coordinating motif in the catalytic centers of various mononuclear non-heme Fe(II) enzymes. Although these active sites are based on totally unrelated protein folds and bring about a wide range of chemical transformations, most of them share the ability to couple dioxygen reduction with the oxygenation of an organic substrate. With the increasing number of protein structures now solved, it has become clear that the 2-His-1-carboxylate signature is less of a paradigm for non-heme Fe(II) active sites than had long been thought and that it can be replaced by alternative metal centers in various oxygenases, the structure-function relationships and proposed catalytic mechanisms of which are reviewed here. Metal coordination through three histidines and one glutamate constitutes the classical motif described for enzyme members of the cupin protein superfamily, such as aci-reductone dioxygenase and quercetin dioxygenase, multiple metal forms of which (including the Fe(II) type) are found in nature. Cysteine dioxygenase and diketone dioxygenase, which are strictly Fe(II)-dependent oxygenases based on the cupin fold, bind the catalytic metal through the homologous triad of histidines, but lack the fourth glutamate ligand. An alpha-ketoglutarate-dependent Fe(II) halogenase shows metal coordination by two histidines as the only protein-derived ligands, whilst carotene oxygenase, from a different protein fold family, features an Fe(II) site consisting of four histidine side chains. These recently discovered metallocenters are discussed with respect to their metal-binding properties and the reaction coordinates of the O(2)-dependent conversions they catalyze.     

Refinement of protein cores and protein-peptide interfaces using a potential scaling approach

Refinement of side chain conformations in protein model structures and at the interface of predicted protein-protein or protein-peptide complexes is an important step during protein structural modelling and docking. A common approach for side chain prediction is to assume a rigid protein main chain for both docking partners and search for an optimal set of side chain rotamers to optimize the steric fit. However, depending on the target-template similarity in the case of comparative protein modelling and on the accuracy of an initially docked complex, the main chain template structure is only an approximation of a realistic target main chain. An inaccurate rigid main chain conformation can in turn interfere with the prediction of side chain conformations. In the present study, a potential scaling approach (PS-MD) during a molecular dynamics (MD) simulation that also allows the inclusion of explicit solvent has been used to predict side chain conformations on semi-flexible protein main chains. The PS-MD method converges much faster to realistic protein-peptide interface structures or protein core structures than standard MD simulations. Depending on the accuracy of the protein main chain, it also gives significantly better results compared with the standard rotamer search method.     

Protein folds and protein folding

The classification of protein folds is necessarily based on the structural elements that distinguish domains. Classification of protein domains consists of two problems: the partition of structures into domains and the classification of domains into sets of similar structures (or folds). Although similar topologies may arise by convergent evolution, the similarity of their respective folding pathways is unknown. The discovery and the characterization of the majority of protein folds will be followed by a similar enumeration of available protein folding pathways. Consequently, understanding the intricacies of structural domains is necessary to understanding their collective folding pathways. We review the current state of the art in the field of protein domain classification and discuss methods for the systematic and comprehensive study of protein folding across protein fold space via atomistic molecular dynamics simulation. Finally, we discuss our large-scale Dynameomics project, which includes simulations of representatives of all autonomous protein folds.     

Favorable scaffolds: proteins with different sequence, structure and function may associate in similar ways

Proteins with similar structures may have different functions. Here, using a non-redundant two-chain protein-protein interface dataset containing 103 clusters, we show that this paradigm extends to interfaces. Whereas usually similar interfaces are obtained from globally similar chains, this is not always the case. Remarkably, in some interface clusters, although the interfaces are similar, the overall structures and functions of the chains are different. Hence, our work suggests that different folds may combinatorially assemble to yield similar local interface motifs. The preference of different folds to associate in similar ways illustrates that the paradigm is universal, whether for single chains in folding or for protein-protein association in binding. We analyze and compare the two types of clusters. Type I, with similar interfaces, similar global structures and similar functions, is better packed, less planar, has larger total and non-polar buried surface areas, better complementarity and more backbone-backbone hydrogen bonds than Type II (similar interfaces, different global structures and different functions). The dataset clusters may provide rich data for protein-protein recognition, cellular networks and drug design. In particular, they should be useful in addressing the difficult question of what the favorable ways for proteins to interact are.     

Stereochemical analysis of acrylic copolymers with large polar side groups by NMR spectroscopy

NMR spectra give abundant information at the microstructural level. When pseudoasymmetric carbon atoms are present in the monomeric repeating units, it is possible to obtain valuable information about the relative stereochemical configuration of the side substituents of the pseudoasymmetric center. This is the best way to know the tacticity of a polymer and the cotacticity parameters of copolymers, as well as the average distribution of comonomeric units along the chains, according to the polymerization mechanism. In this work, we present the microstructural and stereochemical characterization of acrylic copolymers prepared by free radical copolymerization of 2-hydroxyethyl methacrylate with a methacrylic ester or methacrylamide derivative, bearing polar side groups of biomedical interest. These copolymer systems were analyzed by ¹H-NMR spectroscopy to determine the average molar composition, and the corresponding reactivity ratios, and by ¹³C-NMR to study the microstructural and stereochemical configuration of comonomer sequences in terms of triads and pentads. Analyses account for the mechanism of polymerization, which fits first order Markov statistics for the addition of comonomers to free radical growing ends. The stereochemical distribution is described by a Bernoullian trial.     

Closed loops: persistence of the protein chain returns

It has recently been discovered that globular proteins are universallybuilt from standard loop-n-lock units of about 30 amino acidresidues. The hypothesis has been put forward on the loop stagein the protein evolution when the units were autonomous. Laterthey joined together making longer chains. One would expectthat the early individual loop-n-lock elements might still bedetected in modern protein sequences as remnants of the hypothetical30-residue sequence prototypes. Among several strong sequencemotifs, extracted from protein sequences of 23 complete bacterialproteomes, one 32-residue prototype was studied here in detail.Numerous sequence segments related to the prototype are identifiedin the crystal structures of proteins of a PDB_SELECT database.Analysis of the respective chain trajectories for the caseswith different degrees of sequence conservation confirms thatthe majority of the segments correspond to the closed loops.In the evolutionary diversification of the prototypes the secondarystructure yields first, while the sequence is still moderatelyconserved. The last feature to go is the chain return property.Apparently, the opening of the loops would severely destabilizethe protein fold, which explains their conservation.