Structural studies of spider silk proteins in the fiber

Although spider silk has been studied for a number of years the structures of the proteins involved have yet to be definitely determined. X-ray diffraction and solid-state nuclear magnetic resonance (NMR) were used to study major ampullate (dragline) silk from Nephila clavipes. The silk was studied in its natural state, in the supercontacted state and in the restretched state following supercontraction. The natural silk structure is dominated by β-sheets aligned parallel to the fiber axis. Supercontraction is characterized by randomizing of the orientation of the β-sheet. When the fiber is restretched alignment is regained. However, the same reorientation was observed for wetting of minor ampullate silk which does not supercontract. Thus, the reorientation of β-sheets alone cannot explain the supercontraction in dragline silk. Cocoon silk showed very little β-sheet orientation in the natural state and there were no changes upon wetting. NMR and X-ray diffraction data are consistent with the β-sheets arising from the poly-alanine sequences known to be present in the proteins of major ampullate silk as has been proposed previously. © 1997 John Wiley & Sons, Ltd.     

A structural view on spider silk proteins and their role in fiber assembly

Spider silk is the toughest known biomaterial and even outrivals modern synthetic high‐performance materials. The question of understanding fiber formation is how the spider can prevent premature and fatal aggregation processes inside its own body and how the chemical and mechanical stimuli used to induce the fiber formation process translate into structural changes of the silk material, finally leading to controlled and irreversible aggregation. Here, the focus will be on the structure and function of the highly conserved N‐domains and C‐terminal domains of spider dragline silk which, unlike the very long repetitive sequence elements, adopt a folded conformation in solution and are therefore able to control intermolecular interactions and aggregation between other spider silk molecules. The structures of these domains add valuable details for the construction of a molecular picture of the complicated and highly optimized silk assembly process that might be beneficial for large‐scale in vitro fiber formation attempts with recombinant silk material. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The dimerization mechanism of the N-terminal domain of spider silk proteins is conserved despite extensive sequence divergence

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.     

Temperature-dependent electrical resistivity of the hornet cuticle: Changes induced by colchicine and purine in the diet

Various cuticular areas of the Oriental hornet (Vespa orientalis) were measured for their electrical resistivity within the range of 3–40°C. It was found that: (1) the electrical resistivity is temperature dependent; (2) there are no significant differences in this respect between hornets collected in different regions; (3) there are no differences in the electrical resisitivity between the yellow and brown areas of the cuticle in control hornets; (4) there are however, significant changes in resistance between the yellow and brown strips of hornets fed on purine; (5) hornets fed on colchicine reach a minimum resistance at a higher temperature than do the control hornets; and (6) hornets fed on purine reach a minimum resistance at a lower temperature. It is suggested that the cuticular changes in resistivity at different temperatures reflect the hornets' mechanism of detecting and adjusting to changes in the ambient temperature.     

Conformation of alpha zeins in solid state by Fourier transform IR

The major maize storage proteins (alpha zeins) are deposited as an insoluble mass in the protein bodies of the endosperm. Because they are insoluble in water, most structural studies are performed in alcohol solutions. To solve the question raised by several authors about denaturation of the alpha zein structure by alcohol, we analyze the secondary structure of alpha zeins prepared with and without solubilization in alcohol (corn gluten meal and protein bodies with high concentrations of alpha zeins and traces of beta zeins). The secondary structures of alpha zeins are analyzed in the solid state by Fourier transform IR spectroscopy (FTIR) in KBr pellets and solid-state 13C-NMR spectroscopy. The proportion of secondary structures obtained by FTIR of alpha zeins prepared with and without solubilization in alcohol yield almost identical proportions of alpha helices and beta sheets. The proportion of alpha helices (43%) agrees with that measured by circular dichroism in an alcohol solution. However, the proportion of beta sheets (28%) is higher than the one measured by the same technique. Gluten and protein body samples with high beta zein content showed higher beta sheet and lower alpha helix proportions than that obtained for alpha zein preparations. The solid-state 13C-NMR spectra show the carbonyl peak for the alpha zeins at delta 176 and for the sample rich in beta zeins at delta 172, which demonstrates the presence of a high content of alpha helices and beta sheets, respectively. These results indicate that alcohol solubilization does not affect the conformation of alpha zeins, validating the secondary structure measurements in solution.     

丝蛋白合成和分泌期家蚕丝腺中有氧氧化特性分析

【目的】有氧氧化中葡萄糖(Glc)、丙酮酸(PA)、乙酰Co A(AC)、还原型吡啶核苷酸(NADH)和腺苷三磷酸(ATP)摩尔数的理论比值为1∶2∶2∶10∶30~32,而己糖激酶(HK)、磷酸果糖激酶1(PFK1)、丙酮酸激酶(PK)、丙酮酸脱氢酶(PDH)、柠檬酸合酶(CS)、异柠檬酸脱氢酶(ICDHm)、α-酮戊二酸脱氢酶(α-KGDH)、NADH泛醌还原酶(NCR)、琥珀酸泛醌还原酶(SCR)、泛醌细胞色素C还原酶(CCR)、细胞色素C氧化酶(CCO)和ATP合酶(AS)活性的理论比值为1∶1∶2∶2∶2∶2∶2∶10∶2∶12∶12∶26~28。本研究旨在分析丝蛋白合成和分泌期家蚕Bombyx mori丝腺有氧氧化的特性。【方法】利用分光光度法和高效液相色谱法测定了上述生化指标的变化。【结果】丝蛋白合成和分泌期家蚕丝腺中检测不到Glc,产物含量以1/30 ATP,1/10 NADH,1/2 AC和1/2 PA的顺序递增;糖酵解途径相关酶活性,以PFK1活性最低;三羧酸循环相关酶活性以1/2 ICDHm,1/2α-KGDH和1/2 CS的顺序递增;氧化磷酸化相关酶包括1/26 AS,1/10 NCR,1/2 SCR,1/12 CCR和1/12 CCO的活性以1/26 AS活性最低;1/26 AS,1/2 ICDHm,1/2 PDH和PFK1的活性依次递增。NADH含量、ATP含量、PFK1活性、PDH活性和NCR活性在丝蛋白合成期升高,但在丝蛋白分泌期下降。【结论】据此推测,家蚕丝腺中PFK1,ICDHm和AS分别是糖酵解途径、三羧酸循环和氧化磷酸化的限速酶;糖酵解途径、丙酮酸脱氢、三羧酸循环和氧化磷酸化速率依次递减;有氧氧化速率在丝蛋白合成期升高,相反在丝蛋白分泌期降低。     

Low pressure‐induced secondary structure transitions of regenerated silk fibroin in its wet film studied by time‐resolved infrared spectroscopy

The secondary structure transitions of regenerated silk fibroin (RSF) under different external perturbations have been studied extensively, except for pressure. In this work, time‐resolved infrared spectroscopy with the attenuated total reflectance (ATR) accessory was employed to follow the secondary structure transitions of RSF in its wet film under low pressure. It has been found that pressure alone is favorable only to the formation of β‐sheet structure. Under constant pressure there is an optimum amount of D₂O in the wet film (D₂O : film = 2:1) so as to provide the optimal condition for the reorganization of the secondary structure and to have the largest formation of β‐sheet structure. Under constant amount of D₂O and constant pressure, the secondary structure transitions of RSF in its wet film can be divided into three stages along with time. In the first stage, random coil, α‐helix, and β‐turn were quickly transformed into β‐sheet. In the second stage, random coil and β‐turn were relatively slowly transformed into β‐sheet and α‐helix, and the content of α‐helix was recovered to the value prior to the application of pressure. In the third and final stage, no measurable changes can be found for each secondary structure. This study may be helpful to understand the secondary structure changes of silk fibroin in silkworm's glands under hydrostatic pressure.     

Analyses of circular dichroism spectra of membrane proteins

Circular dichroism (CD) spectroscopy is a valuable technique for the determination of protein secondary structures. Many linear and nonlinear algorithms have been developed for the empirical analysis of CD data, using reference databases derived from proteins of known structures. To date, the reference databases used by the various algorithms have all been derived from the spectra of soluble proteins. When applied to the analysis of soluble protein spectra, these methods generally produce calculated secondary structures that correspond well with crystallographic structures. In this study, however, it was shown that when applied to membrane protein spectra, the resulting calculations produce considerably poorer results. One source of this discrepancy may be the altered spectral peak positions (wavelength shifts) of membrane proteins due to the different dielectric of the membrane environment relative to that of water. These results have important consequences for studies that seek to use the existing soluble protein reference databases for the analyses of membrane proteins.     

Analyses of the general rule on residue pair frequencies in local amino acid sequences of soluble,ordered proteins

The amino acid sequences of soluble, ordered proteins with stable structures have evolved due to biological and physical requirements, thus distinguishing them from random sequences. Previous analyses have focused on extracting the features that frequently appear in protein substructures, such as α‐helix and β‐sheet, but the universal features of protein sequences have not been addressed. To clarify the differences between native protein sequences and random sequences, we analyzed 7368 soluble, ordered protein sequences, by inspecting the observed and expected occurrences of 400 amino acid pairs in local proximity, up to 10 residues along the sequence in comparison with their expected occurrence in random sequence. We found the trend that the hydrophobic residue pairs and the polar residue pairs are significantly decreased, whereas the pairs between a hydrophobic residue and a polar residue are increased. This trend was universally observed regardless of the secondary structure content but was not observed in protein sequences that include intrinsically disordered regions, indicating that it can be a general rule of protein foldability. The possible benefits of this rule are discussed from the viewpoints of protein aggregation and disorder, which are both caused by low‐complexity regions of hydrophobic or polar residues.     

A 3D building blocks approach to analyzing and predicting structure of proteins

A new approach is introduced for analyzing and ultimately predicting protein structures, defined at the level of C alpha coordinates. We analyze hexamers (oligopeptides of six amino acid residues) and show that their structure tends to concentrate in specific clusters rather than vary continuously. Thus, we can use a limited set of standard structural building blocks taken from these clusters as representatives of the repertoire of observed hexamers. We demonstrate that protein structures can be approximated by concatenating such building blocks. We have identified about 100 building blocks by applying clustering algorithms, and have shown that they can "replace" about 76% of all hexamers in well-refined known proteins with an error of less than 1 A, and can be joined together to cover 99% of the residues. After replacing each hexamer by a standard building block with similar conformation, we can approximately reconstruct the actual structure by smoothly joining the overlapping building blocks into a full protein. The reconstructed structures show, in most cases, high resemblance to the original structure, although using a limited number of building blocks and local criteria of concatenating them is not likely to produce a very precise global match. Since these building blocks reflect, in many cases, some sequence dependency, it may be possible to use the results of this study as a basis for a protein structure prediction procedure.     

Characterization of the amino acid contribution to the folding degree of proteins

The folding degree index (Estrada, Bioinformatics 2002;18:697-704) is extended to account for the contribution of amino acids to folding. First, the mathematical formalism for extending the folding degree index is presented. Then, the amino acid contributions to folding degree of several proteins are used to analyze its relation to secondary structure. The possibilities of using these contributions in helping or checking the assignation of secondary structure to amino acids are also introduced. The influence of external factors to the amino acids contribution to folding degree is studied through the temperature effect on ribonuclease A. Finally, the analysis of 3D protein similarity through the use of amino acid contributions to folding degree is studied by selecting a series of lysozymes. These results are compared to that obtained by sequence alignment (2D similarity) and 3D superposition of the structures, showing the uniqueness of the current approach.     

An automatic method involving cluster analysis of secondary structures for the identification of domains in proteins.

With a growing number of structures available in the Brookhaven Protein Data Bank, automatic methods for domain identification are required for the construction of databases. Domains are considered to be clusters of secondary structure elements. Thus, helices and strands are first clustered using intersecondary structural distances between C alpha positions, and dendrograms based on this distance measure are used to identify domains. Individual domains are recognized by a disjoint factor, which enables the automatic identification and classification into disjoint, interacting, and conjoint domains. Application to a database of 83 protein families and 18 unique structures shows that the approach provides an effective delineation of boundaries and identifies those proteins that can be considered as a single domain. A quantitative estimate of the interaction between domains has been proposed. The database of protein domains is a useful tool for understanding protein folding, for recognizing protein folds, and for understanding structure-activity relationships.     

Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold.

The solution conformation of three peptides corresponding to the two beta-hairpins and the alpha-helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non-native locally folded structures, but no well-defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the alpha-helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non-helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native-like secondary structure propensities, although a minimum tendency to avoid non-native structures and to favor native ones could be required.     

A new perspective on analysis of helix-helix packing preferences in globular proteins

For many years, statistical analysis of protein databanks has led to the belief that the steric compatibility of helix interfaces may be the source of observed preferences for particular angles between neighboring helices. Several elegant models describing how side chains on helices can interdigitate without steric clashes were able to account quite reasonably for the observed distributions. However, it was later recognized that the 'bare' measured angle distribution should be corrected to avoid statistical bias.12 Disappointingly, the rescaled distributions dramatically lost their similarity with theoretical predictions, casting doubts on the validity of the geometrical assumptions and models. In this article, we elucidate a few points concerning the proper choice of a random reference distribution. In particular we demonstrate the need for corrections induced by unavoidable uncertainties in determining whether two helices are in face-to-face contact or not and their relative orientations. By using this new rescaling, we show that 'true' packing angle preferences are well described by regular packing models, thus proving that preferential angles between contacting helices do exist.     

Description and recognition of regular and distorted secondary structures in proteins using the automated protein structure analysis method

The Automated Protein Structure Analysis (APSA) method, which describes the protein backbone as a smooth line in three‐dimensional space and characterizes it by curvature κ and torsion τ as a function of arc length s, was applied on 77 proteins to determine all secondary structural units via specific κ(s) and τ(s) patterns. A total of 533 α‐helices and 644 β‐strands were recognized by APSA, whereas DSSP gives 536 and 651 units, respectively. Kinks and distortions were quantified and the boundaries (entry and exit) of secondary structures were classified. Similarity between proteins can be easily quantified using APSA, as was demonstrated for the roll architecture of proteins ubiquitin and spinach ferridoxin. A twenty‐by‐twenty comparison of all α domains showed that the curvature‐torsion patterns generated by APSA provide an accurate and meaningful similarity measurement for secondary, super secondary, and tertiary protein structure. APSA is shown to accurately reflect the conformation of the backbone effectively reducing three‐dimensional structure information to two‐dimensional representations that are easy to interpret and understand. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Diversity of functions of proteins with internal symmetry in spatial arrangement of secondary structural elements.

We carry out a systematic analysis of the correlation between similarity of protein three-dimensional structures and their evolutionary relationships. The structural similarity is quantitatively identified by an all-against-all comparison of the spatial arrangement of secondary structural elements in nonredundant 967 representative proteins, and the evolutionary relationship is judged according to the definition of superfamily in the SCOP database. We find the following symmetry rule: a protein pair that has similar folds but belong to different superfamilies has (with a very rare exception) certain internal symmetry in its common similar folds. Possible reasons behind the symmetry rule are discussed.     

Prediction of the three-dimensional structure of proteins using the electrostatic screening model and hierarchic condensation

We describe a method for predicting the three-dimensional (3-D) structure of proteins from their sequence alone. The method is based on the electrostatic screening model for the stability of the protein main-chain conformation. The free energy of a protein as a function of its conformation is obtained from the potentials of mean force analysis of high-resolution x-ray protein structures. The free energy function is simple and contains only 44 fitted coefficients. The minimization of the free energy is performed by the torsion space Monte Carlo procedure using the concept of hierarchic condensation. The Monte Carlo minimization procedure is applied to predict the secondary, super-secondary, and native 3-D structures of 12 proteins with 28–110 amino acids. The 3-D structures of the majority of local secondary and super-secondary structures are predicted accurately. This result suggests that control in forming the native-like local structure is distributed along the entire protein sequence. The native 3-D structure is predicted correctly for 3 of 12 proteins composed mainly from the α-helices. The method fails to predict the native 3-D structure of proteins with a predominantly β secondary structure. We suggest that the hierarchic condensation is not an appropriate procedure for simulating the folding of proteins made up primarily from β-strands. The method has been proved accurate in predicting the local secondary and super-secondary structures in the blind ab initio 3-D prediction experiment. Proteins 31:74–96, 1998. © 1998 Wiley-Liss, Inc.     

Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regression

The secondary structures of proteins (alpha-helical, beta-sheet, beta-turn, and random coil) in the solid state and when bound to polymer beads, containing immobilized phenyl and butyl ligands such as those as commonly employed in hydrophobic interaction chromatography, have been investigated using FTIR-ATR spectroscopy and partial least squares (PLS) methods. Proteins with known structural features were used as models, including 12 proteins in the solid state and 7 proteins adsorbed onto the hydrophobic surfaces. A strong PLS correlation was achieved between predictions derived from the experimental data for 4 proteins adsorbed onto the phenyl-modified beads and reference data obtained from the X-ray crystallographic structures with r(2) values of 0.9974, 0.9864, 0.9924, and 0.9743 for alpha-helical, beta-sheet, beta-turn, and random coiled structures, respectively. On the other hand, proteins adsorbed onto the butyl sorbent underwent greater secondary structural changes compared to the phenyl sorbent as evidenced from the poorer PLS r(2) values (r(2) are 0.9658, 0.9106, 0.9571, and 0.9340). The results thus indicate that the secondary structures for these proteins were more affected by the butyl sorbent, whereas the secondary structure remains relatively unchanged for the proteins adsorbed onto the phenyl sorbent. This study has important ramifications for understanding the nature of protein secondary structural changes following adsorption onto hydrophobic sorbent surfaces. This knowledge could also enable the development of useful protocols for enhancing the chromatographic purification of proteins in their native bioactive states. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 895-905, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

重组杂合蛛丝蛋白MiSpNT-PySpRp-MiSpCT的二级结构表征

重组蛛丝纤维作为一种性能优异的生物材料,具有良好的生物相容性,在生物医学工程领域具有极大的潜在应用价值。已有研究表明,重组蛛丝蛋白可用作血管、神经导管及药物载体等,但其生物学功能仍有待研究。本研究以大腹园蛛基因组为模板,设计特异性引物,通过PCR扩增获得大腹园蛛梨状腺丝（piriform spidroin: PySp）一个完整重复区（Rp）编码序列;此Rp模块与MiSpNT/CT模块重组,构建微小型杂合蛛丝蛋白MiSpNT-PySpRp-MiSpCT,成功在大肠杆菌BL21中高效表达,借助8 mol/L尿素裂解缓冲液进行变性纯化,得到纯度较高的杂合蛛丝蛋白MiSpNT-PySpRp-MiSpCT,产量约100 mg/L。CD图谱显示,MiSpNT-PySpRp-MiSpCT蛋白质溶液主要以α-螺旋和无规卷曲形式存在,随着溶液pH值降低,部分α-螺旋向β-折叠转变;红外光谱显示,在自然成丝及冻干过程中,部分α-螺旋转化为β-折叠,符合天然蛛丝蛋白成丝过程的二级结构变化特征。本研究结果为今后获得具有天然蛛丝纤维优异性能的人工重组蛛丝纤维材料提供一种新的可能。