Fine structure of a membrane anchor domain

We describe a detailed deletion analysis of the anchoring domain of a model membrane protein. Removal of the 23 contiguous uncharged amino acids from the carboxy terminus of the bacteriophage fl gene III protein (pIII) converts it from an integral membrane protein to a secreted periplasmic form. Deletions that remove six or fewer residues of the hydrophobic core result in no diminution of the protein's capacity to anchor in the membrane. Longer deletions into this hydrophobic domain gradually destablize the protein-membrane association. pIII derivatives with over half of the hydrophobic core deleted retain substantial residual anchor function. The basic residues, arginine and lysine, which provide a carboxy-terminal boundary for this domain, can be deleted without loss of anchoring capacity.     

Study and prediction of secondary structure for membrane proteins

In this paper we present a novel approach to membrane protein secondary structure prediction based on the statistical stepwise discriminant analysis method. A new aspect of our approach is the possibility to derive physical-chemical properties that may affect the formation of membrane protein secondary structure. The certain physical-chemical properties of protein chains can be used to clarify the formation of the secondary structure types under consideration. Another aspect of our approach is that the results of multiple sequence alignment, or the other kinds of sequence alignment, are not used in the frame of the method. Using our approach, we predicted the formation of three main secondary structure types (alpha-helix, beta-structure and coil) with high accuracy, that is Q(3) = 76%. Predicting the formation of alpha-helix and non-alpha-helix states we reached the accuracy which was measured as Q(2) = 86%. Also we have identified certain protein chain properties that affect the formation of membrane protein secondary structure. These protein properties include hydrophobic properties of amino acid residues, presence of Gly, Ala and Val amino acids, and the location of protein chain end.     

Detergents for the stabilization and crystallization of membrane proteins

The use of detergents for the structural study of membrane proteins is discussed with an emphasis on practical issues relating to membrane solubilization, protein aggregation, detergent purity and detergent quantitation. Detergents are useful reagents as mimics of lipid bilayers because of their self-assembling properties, but as a result, they have complex properties in solution. It can be difficult to maintain a solubilized membrane protein in a native conformational state, and the non-specific aggregation of detergent-solubilized proteins is a common problem. Empirical "stability screens" can be helpful in choosing which detergents, and which detergent concentrations, may be optimal for a given system.     

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.     

Model structure of the prototypical non-fimbrial adhesin YadA of Yersinia enterocolitica

Non-fimbrial adhesins, such as Yersinia YadA, Moraxella UspA1 and A2, Haemophilus Hia and Hsf, or Bartonella BadA represent an important class of molecules by which pathogenic proteobacteria adhere to their hosts. They form trimeric surface structures with a head-stalk-anchor architecture. Whereas head and stalk domains are diverse and appear (frequently repetitively) in different combinations, the anchor domains are homologous and display the properties of autotransporters. We have built a molecular model for the prototypical non-fimbrial adhesin, YadA, by combining the crystal structure of the head (PDB:1P9H) with theoretical models for the stalk and the anchor. The head domain is a single-stranded, left-handed beta-helix, connected to the stalk by a conserved trimerization element (the neck). The stalk consists of a right-handed coiled coil, containing ten 15-residue repeats with a C-terminal stutter (insertion of four residues). The stalk continues into the conserved anchor domain, which is formed by four heptads of a left-handed coiled coil, followed by four transmembrane beta-strands. Our model of the YadA coiled coil, generated with the program BeammotifCC, combines these periodicities into a structure that starts with a pronounced right-handed supercoil and ends with a canonical, left-handed conformation. The last two heptads of the coiled coil are located within a 12-stranded beta-barrel, formed by trimerization of the four transmembrane beta-strands in each monomer. We propose that this pore assembles in the outer membrane to form the opening through which the monomer chains exit the cell. After export is completed, the fiber folds and the pore is occluded by the coiled coil. Our model explains how these proteins can act as autotransporters in the absence of any homology to classical, single-chain autotransporters.     

Spectroscopic methods for analysis of protein secondary structure

Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of secondary structures in proteins. NMR chemical shifts may also be used to determine the positions of secondary structure within the primary sequence of a protein. However, the chemical shifts must first be assigned to particular residues, making the technique considerably slower than the optical methods. These data, together with sophisticated molecular modeling techniques, allow for refinement of protein structural models as well as rapid assessment of conformational changes resulting from ligand binding or macromolecular interactions. A selected number of examples are given to illustrate the power of the techniques in applications of biological interest.     

Predicted secondary structure and membrane topology of the scrapie prion protein

The integral membrane sialoglycoprotein PrPSc is the only identifiable component of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakob disease in humans are transmissible, degenerative neurological diseases caused by prions. Standard predictive strategies have been used to analyze the secondary structure of the prion protein in conjunction with Fourier analysis of the primary sequence hydrophobicities to detect potential amphipathic regions. Several hydrophobic segments, a proline- and glycine-rich repeat region and putative glycosylation sites are incorporated into a model for the integral membrane topology of PrP. The complete amino acid sequences of the hamster, human and mouse prion proteins are compared and the effects of residue substitutions upon the predicted conformation of the polypeptide chain are discussed. While PrP has a unique primary structure, its predicted secondary structure shares some interesting features with the serum amyloid A proteins. These proteins undergo a post-translational modification to yield amyloid A, molecules that share with PrP the ability to polymerize into birefringent filaments. Our analyses may explain some experimental observations on PrP, and suggest further studies on the properties of the scrapie and cellular PrP isoforms.     

Purification of a membrane glycoprotein with an inositol-containing phospholipid anchor from Dictyostelium discoideum.

Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl beta-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 x 10(10) cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.     

Emerging functional roles for the glycosyl-phosphatidylinositol membrane protein anchor

Conclusion Experimental evidence has accumulated over the past few years to suggest that the GPI protein anchor may play a broad role in the regulation of membrane protein function. The significant changes in the biophysical properties of proteins that are membrane-anchored through GPI in lieu of a hydrophobic transmembrane peptide indicates a variety phobic transmembrane peptide indicates a variety of potential new functions served by the anchor structure itself. Moreover, the number of structural variations within the family of GPI molecules indicates a further opportunity for subspecialization of such anchored proteins, especially regarding cellular localization, mobility, metabolism and susceptibility to enzymatically-induced release. It is likely that further exploration of the structure and function of the GPI anchor may reveal additional roles for this unusual mechanism of membrane-protein attachment.     

淫羊藿多糖的分离纯化及结构初步分析

从淫羊藿中提取多糖并鉴定其初步结构和单糖组成.采用超声-水提醇沉法提取粗多糖、Sevag法去蛋白、DEAE-52纤维素及Sephadex G-100柱层析法纯化得到淫羊藿多糖EPSⅠ-1和EPSⅡ-1.应用紫外光谱法和红外光谱法对其结构做初步分析.采用高效液相色谱法(HPLC)测定其单糖组成及摩尔比.均一的EPSⅠ-1和EPSⅡ-1多糖在紫外和红外中具有多糖的特征吸收峰,组成中含有吡喃环结构;EPSⅠ-1的单糖组成为鼠李糖和葡萄糖,摩尔比为1:1.13;EPSⅡ-1的单糖组成为果糖、葡萄糖和一个不确定的糖,摩尔比为1:1.91.有效地分离纯化了淫羊藿多糖,这为淫羊藿多糖的广泛应用奠定了实验基础.     

Solution structure of the glycosylphosphatidylinositol membrane anchor glycan of Trypanosoma brucei variant surface glycoprotein

The average solution conformation of the glycosylphosphatidylinositol (GPI) membrane anchor of Trypanosoma brucei variant surface glycoprotein (VSG) has been determined by using a combination of two-dimensional 1H-1H NMR methods together with molecular orbital calculations and restrained molecular dynamics simulations. This allows the generation of a model to describe the orientation of the glycan with respect to the membrane. This shows that the glycan exists in an extended configuration along the plane of the membrane and spans an area of 600 A2, which is similar to the cross-sectional area of a monomeric N-terminal VSG domain. Taken together, these observations suggest a possible space-filling role for the GPI anchor that may maintain the integrity of the VSG coat. The potential importance of the GPI glycan as a chemotherapeutic target is discussed in light of these observations.     

Purification and crystallization of the EcoRV restriction endonuclease

The type II restriction endonuclease EcoRV purified from a genetically engineered, overproducing strain has been crystallized. Four crystal forms all obtained by precipitation with polyethylene glycol 4000 have been characterized. Two of these are suitable for high resolution structure analysis. Both are orthorhombic, have space group P2(1)2(1)2(1) and have similar unit cell dimensions of a = 58.2 A, b = 71.7 A, c = 130.6 A (form A) and a = 59.9 A, b = 74.5 A, c = 121.8 A (form B). They diffract to about 2A resolution and appear to have one dimer of 2 X 29,000 daltons in the asymmetric unit.     

Purification and secondary structure determination of simian immunodeficiency virus p27

We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus. Circular dichroism measurements of purified p27 were used to determine the relative amounts of alpha-helix, beta-sheet and unordered secondary structural elements. These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons.     

Spectral decomposition for the search and analysis of RNA secondary structure.

Scales in RNA, based on geometrical considerations, can be exploited for the analysis and prediction of RNA structures. By using spectral decomposition, geometric information that relates to a given RNA fold can be reduced to a single positive scalar number, the second eigenvalue of the Laplacian matrix corresponding to the tree-graph representation of the RNA secondary structure. Along with the free energy of the structure, being the most important scalar number in the prediction of RNA folding by energy minimization methods, the second eigenvalue of the Laplacian matrix can be used as an effective signature for locating a target folded structure given a set of RNA folds. Furthermore, the second eigenvector of the Laplacian matrix can be used to partition large RNA structures into smaller fragments. An illustrative example is given for the use of the second eigenvalue to predict mutations that may cause structural rearrangements, thereby disrupting stable motifs.     

Purification,crystallization, and preliminary X-ray analysis of Bacillus subtilis ferrochelatase

Bacillus subtilis ferrochelatase (EC 4.99.1.1), the final enzyme in protoheme IX biosynthesis, was produced with an inducible T7 RNA polymerase expression system in Escherichia coli and purified from the soluble cell fraction. It was crystallized from polyethylene glycol solution using the microseeding technique. The crystals diffract to a minimum Bragg spacing of 2.1 Å. The space group is P4₂ with unit cell dimensions a = b = 50.2 Å, c = 120.1 Å. © 1995 Wiley-Liss, Inc.