Determining the binding capability of the mouse major urinary proteins using 2-naphthol as a fluorescent probe

The mouse major urinary proteins (MUPs) are an ensemble of isoforms secreted by adult male mice and involved in sexual olfactory communication. MUPs belong to the lipocalin superfamily, whose conserved structure is a beta-barrel made of eight antiparallel beta-strands forming a hydrophobic pocket that accommodates small organic molecules. A detailed knowledge of the molecular mechanism associated to the binding of those molecules can guide protein engineering to devise mutated proteins where the ligand specificity, binding affinity, and release rate can be modulated. Proteins with such peculiar properties may have interesting biotechnological applications for pest control, as well as in food and cosmetic industries. In this work, we demonstrate that the fluorescent molecule 2-naphthol binds to the natural ligand's binding site of MUPs with high affinity. In addition, we show that 2-naphthol binds to MUPs in its protonated form, that its fluorescence is blue-shifted, and the quantum yield is increased, thus confirming the high hydrophobicity of the protein pocket and the absence of proton acceptors inside the binding site. At large the results presented, besides demonstrating that the use of 2-naphthol provides a convenient and quick method for testing MUPs binding activity and to ascertain the quality of the protein preparation, suggest that MUPs can represent an interesting system for studying the photophysical characteristics of fluorescent molecules in a highly hydrophobic environment.     

A general model of the P2 protein of peripheral nervous system myelin based on secondary structure predictions, tertiary folding principles, and experimental observations

The amino acid sequence of the P2 protein of peripheral myelin was analyzed with regard to regions of probable alpha-helix, beta-structure, beta-turn, and unordered conformation by means of several algorithms commonly used to predict secondary structure in proteins. Because of the high beta-sheet content and virtual absence of alpha-helix shown by the circular dichroic spectra of the protein, a bias was introduced into the algorithms to favor the beta-structure over the alpha-helical conformation. In order to define those beta-sheet residues that could lie on the external hydrophilic surface of the protein and those that could lie in its hydrophobic interior, the predicted beta-strands were examined for charged and uncharged amino acids located at alternating positions in the sequence. The sequential beta-strands in the predicted secondary structure were then ordered into beta-sheets and aligned according to generally accepted tertiary folding principles and certain chemical properties peculiar to the P2 protein. The general model of the P2 protein that emerged was a "Greek key" beta-barrel, consisting of eight antiparallel beta-strands with a two-stranded ribbon of antiparallel beta-structure emerging from one end. The model has an uncharged, hydrophobic core and a highly hydrophilic surface. The two Cys residues, which form a disulfide, occur in a loop connecting two adjacent antiparallel strands. Two hydrophilic loops, each containing a cluster of acidic residues and a single Phe, protrude from one end of the molecule. The general model is consistent with many of the properties of the actual protein, including the relatively weak nature of its association with myelin lipids and the positions of amino acid substitutions. Alternative beta-strand orderings yield three specific models having different interstrand connections across the barrel ends.     

Lipocalins as a scaffold

The concept of scaffolds that can be equipped with artificial biochemically active sites has gained recent interest in the field of protein design. Members of the lipocalin protein family represent promising model systems in this respect. Especially prototypic lipocalins, such as the retinol-binding protein or the bilin-binding protein (BBP), exhibit a structurally simple one-domain fold with a conformationally well conserved beta-barrel as their central motif. This type of supersecondary structure is made of a cylindrically closed beta-sheet of eight antiparallel strands. At the open end of the barrel the beta-strands are connected by four loops in a pairwise manner so that a pocket for the ligand is formed. In a rational protein design study a metal-binding site was functionally grafted on the solvent-exposed surface of the beta-barrel, whereby the rigid backbone conformation permitted the spatially defined arrangement of three His side chains. In a combinatorial protein design approach, the natural ligand pocket of a lipocalin was reshaped. In this manner variants of the BBP were engineered which exhibit high affinity and remarkable specificity for haptens like fluorescein and digoxigenin. The so-called 'anticalins', i.e. artificial lipocalins recognizing prescribed ligands, could provide an interesting alternative to recombinant antibody fragments. Consequently, the use of lipocalins as a scaffold opens new applications for members of this functionally diverse protein family in biotechnology and medicine.     

X-ray structure determination of telokin, the C-terminal domain of myosin light chain kinase, at 2.8 A resolution.

The three-dimensional structure of telokin, an acidic protein identical to the C-terminal portion of smooth muscle myosin light chain kinase from turkey gizzard, has been determined at 2.8 A resolution and refined to a crystallographic R-factor of 19.5% for all measured X-ray data from 30 A to 2.8 A. Crystals used in the investigation belonged to the space group P3(2)21, with one molecule per asymmetric unit and unit cell dimensions of a = b = 64.4 A and c = 50.6 A. Telokin contains 154 amino acid residues, 103 of which were visible in the electron density map. The overall molecular fold of telokin consists of seven strands of antiparallel beta-pleated sheet that wrap around to form a barrel. There is also an extended tail of eight amino acid residues at the N terminus that does not participate in beta-sheet formation. The beta-barrel can be simply envisioned as two layers of beta-sheet, nearly parallel to one another, with one layer containing four and the other three beta-strands. This type of beta-barrel, as seen in telokin, was first observed for the CH2 domain of an immunoglobulin fragment Fc. Telokin is an intracellular protein and, as such, does not contain the disulphide linkage between beta-strands B and F normally observed in the immunoglobulin constant domains. It does, however, contain two cysteine amino acid residues (Cys63 and Cys115) that are situated at structurally identical positions to those forming the disulphide linkage in the immunoglobulin constant domain.     

Major urinary proteins, alpha(2U)-globulins and aphrodisin

The major urinary proteins (MUPs) are proteins secreted by the liver and filtered by the kidneys into the urine of adult male mice and rats, the MUPs of rats being also referred to as alpha(2U)-globulins. The MUP family also comprises closely related proteins excreted by exocrine glands of rodents, independently of their sex. The MUP family is an expression of a multi-gene family. There is complex hormonal and tissue-specific regulation of MUP gene expression. The multi-gene family and its outflow are characterized by a polymorphism which extends over species, strains, sexes, and individuals. There is evidence of evolutionary conservation of the genes and their outflow within the species and evidence of change between species. MUPs share the eight-stranded beta-barrel structure lining a hydrophobic pocket, common to lipocalins. There is also a high degree of structural conservation between mouse and rat MUPs. MUPs bind small natural odorant molecules in the hydrophobic pocket with medium affinity in the 10(4)-10(5) M(-1) range, and are excreted in the field, with bound odorants. The odorants are then released slowly in air giving a long lasting olfactory trace to the spot. MUPs seem to play complex roles in chemosensory signalling among rodents, functioning as odorant carriers as well as proteins that prime endocrine reactions in female conspecifics. Aphrodisin is a lipocalin, found in hamster vaginal discharge, which stimulates male copulatory behaviour. Aphrodisin does not seem to bind odorants and no polymorphism has been shown. Both MUPs and aphrodisin stimulate the vomeronasal organ of conspecifics.     

Calorimetric dissection of thermal unfolding of OspA,a predominantly beta-sheet protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from Borrelia burgdorferi is a predominantly beta-sheet protein comprised of beta-strands beta1-beta21 and a short C-terminal alpha-helix. It contains two globular domains (N and C-terminal domains) and a unique single-layer beta-sheet (central beta-sheet) that connects the two domains. OspA contains an unusually large number of charged amino acid residues. To understand the mechanism of stabilization of this unique beta-sheet protein, thorough thermodynamic investigations of OspA and its truncated mutant lacking a part of the C-terminal domain were conducted using calorimetry and circular dichroism. The stability of OspA was found to be sensitive to pH and salt concentration. The heat capacity curve clearly consisted of two components, and all the thermodynamic parameters were obtained for each step. The thermodynamic parameters associated with the two transitions are consistent with a previously proposed model, in which the first transition corresponds to the unfolding of the C-terminal domain and the last two beta-strands of the central beta-sheet, and the second transition corresponds to that of the N-terminal domain and the first beta-strand of the central beta-sheet in the second peak. The ratio of calorimetric and van't Hoff enthalpies indicates that the first peak includes another thermodynamic intermediate state. Large heat capacity changes were observed for both transitions, indicative of large changes in the exposure of hydrophobic surfaces associated with the transitions. This observation demonstrates that hydrophobic parts are buried efficiently in the native structure in spite of the low content of hydrophobic residues in OspA. By decomposing the enthalpy, entropy, and Gibbs free energy into contributions from different interactions, we found that the enthalpy changes for hydrogen bonding and polar interactions are exceptionally large, indicating that OspA maintains its stability by making full use of its unique beta-sheet and high content of polar residues. These thermodynamic analyses demonstrated that it is possible to maintain protein tertiary structure by making effective use of an unusual amino acid composition.     

Consequences of single-site mutations in the intestinal fatty acid binding protein

The intestinal fatty acid binding protein (IFABP) is a small (15 kDa) protein consisting mostly of 10 antiparallel beta-strands (A-J) and a small helical region that serves as a portal for the ligand. Two beta-sheet structures (strands A-E and F-J) surround a cavity into which the ligand binds. In this work, we investigated how changes in the side chains of specific residues are propagated through the structure. To determine what these changes were and how they relate to changes in stability, (15)N chemical shift perturbations were measured and compared to those of the wild-type protein. Seven mutations, five of which change either valine or leucine to glycine, have been examined. All these mutants were less stable than wild-type IFABP, suggesting some structural changes. For five of the mutants, the data suggest that destabilization of a small region of the protein propagates throughout the structure, resulting in an overall decrease in stability. In two (Leu38Gly and Leu89Gly), the loss of cooperativity in the equilibrium denaturation curves suggests that the destabilization of one region may not be transmitted to other regions in a cooperative manner. It is shown that the effect of mutating hydrophobic residues is much greater than that observed upon mutation of a solvent-exposed polar residue.     

Crystallographic analysis of an "anticalin" with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region

The artificial lipocalin FluA with novel specificity toward fluorescein was derived via combinatorial engineering from the bilin-binding protein, BBP by exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal structure of FluA at 2.0 A resolution in the space group P2(1) with two protein-ligand complexes in the asymmetric unit. In both molecules, the characteristic beta-barrel architecture with the attached alpha-helix is well preserved. In contrast, the four loops at one end of the beta-barrel that form the entrance to the binding site exhibit large conformational deviations from the wild-type protein, which can be attributed to the sidechain replacements. Specificity for the new ligand is furnished by hydrophobic packing, charged sidechain environment, and hydrogen bonds with its hydroxyl groups. Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin IX(gamma) in the natural lipocalin. Triggered by the substituted residues, unmutated sidechains at the bottom of the binding site adopt conformations that are quite different from those observed in the BBP, illustrating that not only the loop region but also the hydrophobic interior of the beta-barrel can be reshaped for molecular recognition. Particularly, Trp 129 participates in a tight stacking interaction with the xanthenolone moiety, which may explain the ultrafast electron transfer that occurs on light excitation of the bound fluorescein. These structural findings support our concept of using lipocalins as a scaffold for the engineering of so-called "anticalins" directed against prescribed targets as an alternative to recombinant antibody fragments.     

主要尿蛋白(MUPs)参与化学信息交流和糖脂代谢

主要尿蛋白（MUPs）属于脂质运载蛋白家族,具有保守的中心疏水β链桶状特征性结构域,具有调节种属内与种属间个体间化学信息交流的功能.MUPs主要在肝合成并分泌入血,作为载体与信息素等亲脂性小分子结合,延长其半衰期,一并从肾过滤排泄入尿液,延缓尿迹中信息素的挥发,从而延长信息素的作用时间.啮齿类动物的MUPs本身具有高度多态性,能够发挥类似信息素的作用直接编码个体信息,调节种属内的生物活动.此外,MUPs还能够发挥利它素的功能引起其它种属的畏惧反应.新近研究发现,MUPs受到机体营养状态的调节,与代谢性疾病及糖脂代谢密切相关,但机制尚不清楚.MUPs的功能和机制探索对于化学信息交流与糖脂代谢研究具有重要意义.本文旨在对MUPs的最新研究结果展开简要综述及讨论.     

Crystal structure of D-aminoacylase from Alcaligenes faecalis DA1. A novel subset of amidohydrolases and insights into the enzyme mechanism

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the hydrolysis of N-acyl-D-amino acids. We report here the first D-aminoacylase crystal structure from A. faecalis at 1.5-A resolution. The protein comprises a small beta-barrel, and a catalytic (betaalpha)(8)-barrel with a 63-residue insertion. The enzyme structure shares significant similarity to the alpha/beta-barrel amidohydrolase superfamily, in which the beta-strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One zinc ion is coordinated by Cys(96), His(220), and His(250), while the other is loosely chelated by His(67), His(69), and Cys(96). This is the first example of the metal ion coordination by a cysteine residue in the superfamily. Therefore, D-aminoacylase defines a novel subset and is a mononuclear zinc metalloenzyme but containing a binuclear active site. The preferred substrate was modeled into a hydrophobic pocket, revealing the substrate specificity and enzyme catalysis. The 63-residue insertion containing substrate-interacting residues may act as a gate controlling access to the active site, revealing that the substrate binding would induce a closed conformation to sequester the catalysis from solvent.     

Protein-lipid interactions with Fusobacterium nucleatum major outer membrane protein FomA: spin-label EPR and polarized infrared spectroscopy

FomA, the major outer membrane protein of Fusobacterium nucleatum, was expressed and purified in Escherichia coli and reconstituted from detergent in bilayer membranes of phosphatidylcholines with chain lengths from C(12:0) to C(17:0). The conformation and orientation of membrane-incorporated FomA were determined from polarized, attenuated total reflection, infrared (IR) spectroscopy, and lipid-protein interactions with FomA were characterized by using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled lipids. Approximately 190 residues of membranous FomA are estimated to be in a beta-sheet configuration from IR band fitting, which is consistent with a 14-strand transmembrane beta-barrel structure. IR dichroism of FomA indicates that the beta-strands are tilted by approximately 45 degrees relative to the sheet/barrel axis and that the order parameter of the latter displays a discontinuity corresponding to hydrophobic matching with fluid C(13:0) lipid chains. The stoichiometry ( N b = 23 lipids/monomer) of lipid-protein interaction from EPR demonstrates that FomA is not trimeric in membranes of diC(14:0) phosphatidylcholine and is consistent with a monomeric beta-barrel of 14-16 strands. The pronounced selectivity of interaction found with anionic spin-labeled lipids places basic residues of the protein in the vicinity of the polar-apolar membrane interfaces, consistent with current topology models. Comparison with similar data from the 8- to 22-stranded E. coli outer membrane proteins, OmpA, OmpG, and FhuA, supports the above conclusions.     

Conservation Throughout Vertebrate Evolution of the Predicted β-Strands in Myelin Basic Protein

To identify functionally important parts of the 18.5-kDa myelin basic protein (MBP), the amino acid sequences from 10 species ranging from shark to human were aligned using the SEQHP computer program. The residues that are invariant or very conservatively substituted (Arg/Lys, Ser/Thr, Ile/Leu, Asp/Glu) among all 10 proteins were scored. Of the 72 conserved residues in the 170-residue human protein (42% conserved), 32 are found within the five beta-strands previously predicted (45 residues, 71% conserved), 23 within the small-loops region (42 residues, 55% conserved), but only 17 within the large-loops region (83 residues, 20% conserved). Of the 22 hydrophobic residues within the predicted beta-sheet of human MBP, 20 hydrophobic residues remain in the shark protein, 19 of them in the same positions. In contrast, there are 10 hydrophobic residues elsewhere in the human protein, but only 7 remain in the shark protein and only 1 of them is in the same position. The triprolyl sequence found in all mammalian MBPs and in the chicken MBP is not conserved in the shark protein. The four alternately spliced forms of mouse MBP can be accommodated by the beta-structural model, but not the 17-kDa human MBP, which lacks exon 5. These findings confirm the crucial role of the hydrophobic residues in the predicted beta-sheet for the structure and function of the protein. It seems likely that the conserved portions of the protein make an important contribution to the highly ordered lamellar structure of myelin.     

Deletion variants of Neurospora mitochondrial porin: electrophysiological and spectroscopic analysis

Mitochondrial porins are predicted to traverse the outer membrane as a series of beta-strands, but the precise structure of the resulting beta-barrel has remained elusive. Toward determining the positions of the membrane-spanning segments, a series of small deletions was introduced into several of the predicted beta-strands of the Neurospora crassa porin. Overall, three classes of porin variants were identified: i), those producing large, stable pores, indicating deletions likely outside of beta-strands; ii), those with minimal pore-forming ability, indicating disruptions in key beta-strands or beta-turns; and iii), those that formed small unstable pores with a variety of gating and ion-selectivity properties. The latter class presumably results from a subset of proteins that adopt an alternative barrel structure upon the loss of stabilizing residues. Some variants were not sufficiently stable in detergent for structural analysis; circular dichroism spectropolarimetry of those that were did not reveal significant differences in the overall structural composition among the detergent-solubilized porin variants and the wild-type protein. Several of the variants displayed altered tryptophan fluorescence profiles, indicative of differing microenvironments surrounding these residues. Based on these results, modifications to the existing models for porin structure are proposed.     

Introduction of a lethal redox switch that controls the opening and closing of the hydrophobic cavity in LolA

LolA plays a critical role in the outer membrane sorting of Escherichia coli lipoproteins because it carries a hydrophobic lipoprotein from the inner membrane through the hydrophilic periplasm to the outer membrane receptor LolB. LolA has an incomplete beta-barrel structure composed of 11 beta-strands with an alpha-helical lid forming a hydrophobic cavity inside. The accompanying study revealed that the hydrophobic cavity opens and closes upon the binding and release of lipoproteins, respectively. Ile(93) in the alpha-helix and Phe(140) in the beta-strand are located close to each other in the hydrophobic cavity. These two residues were replaced by Cys to construct the I93C/F140C derivative. Expression of I93C/F140C immediately arrested growth whether wild-type LolA was present or not. However, this dominant negative phenotype was abolished by reducing agents, indicating that the intramolecular disulfide bonding between the two Cys residues is lethal. I93C/F140C was unstable, and its periplasmic level was lower than that of wild-type LolA or its single Cys derivative. Reduction of I93C/F140C was essential for the release of lipoproteins from the inner membrane. Moreover, treatment of I93C/F140C with divalent cross-linkers having different side chain lengths revealed that opening of the lid for a sufficient distance is required for the release activity. The binding of a fluorescent probe to the hydrophobic cavity of I93C/F140C also depended on reducing agents. Taken together, these results indicate that the two Cys residues introduced into LolA function as a redox switch, which regulates the opening and closing of the hydrophobic cavity.     

NMR structure of the integral membrane protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(delta1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel beta-barrel, with successive pairs of beta-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded beta-sheet beyond the height of the barrel. This protruding beta-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.     

Conserved signature proposed for folding in the lipocalin superfamily

We systematically identify a group of evolutionarily conserved residues proposed for folding in a model beta-barrel superfamily, the lipocalins. The nature of conservation at the structural level is defined and we show that the conserved residues are involved in a network of interactions that form the core of the fold. Exploratory kinetic studies are conducted with a model superfamily member, human serum retinol-binding protein, to examine their role. The present results, coupled with key experimental studies conducted with another lipocalin beta-lactoglobulin, suggest that the evolutionarily conserved regions fold on a faster folding time-scale than the non-conserved regions.     

Incorporation of outer membrane protein OmpG in lipid membranes: protein-lipid interactions and beta-barrel orientation

OmpG is an intermediate size, monomeric, outer membrane protein from Escherichia coli, with n beta = 14 beta-strands. It has a large pore that is amenable to modification by protein engineering. The stoichiometry ( N b = 20) and selectivity ( K r = 0.7-1.2) of lipid-protein interaction with OmpG incorporated in dimyristoyl phosphatidylcholine bilayer membranes was determined with various 14-position spin-labeled lipids by using EPR spectroscopy. The limited selectivity for different lipid species is consistent with the disposition of charged residues in the protein. The conformation and orientation (beta-strand tilt and beta-barrel order parameters) of OmpG in disaturated phosphatidylcholines of odd and even chain lengths from C(12:0) to C(17:0) was determined from polarized infrared spectroscopy of the amide I and amide II bands. A discontinuity in the protein orientation (deduced from the beta-barrel order parameters) is observed at the point of hydrophobic matching of the protein with lipid chain length. Compared with smaller (OmpA; n beta = 8) and larger (FhuA; n beta = 22) monomeric E. coli outer membrane proteins, the stoichiometry of motionally restricted lipids increases linearly with the number of beta-strands, the tilt (beta approximately 44 degrees ) of the beta-strands is comparable for the three proteins, and the order parameter of the beta-barrel increases regularly with n beta. These systematic features of the integration of monomeric beta-barrel proteins in lipid membranes could be useful for characterizing outer membrane proteins of unknown structure.     

Chromosomal location, exon/intron organization and evolution of lipocalin genes

Lipocalins exhibit low sequence similarity that contrasts with a tightly conserved folding shared by all members of this superfamily. This conserved folding can be, at least partly, accounted for by a highly conserved gene structure. The array of lipocalin genes that have so far been studied mostly in mammals indicate a large conservation of a typical seven exon/six intron arrangement. Other conserved features include a partly coding exon 1 of variable size, fixed sizes of exons 2-5 that code for an array of lipocalin-specific beta-strands and a tendency of the last exons to either fuse or expand into further exons without major changes in the length of the resulting open reading frame. The conserved exon/intron arrangement as well as a clustering of most lipocalin genes in given chromosomes of human and mouse indicate that the lipocalin genes diverged from a shared ancestor by successive rounds of duplications followed by late changes in exon arrangements.     

Pheromone signalling in the mouse: role of urinary proteins and vomeronasal organ.

The lipocalin protein family is characterized in structure by a conserved hydrophobic pocket which can bind small volatile odorants. The Major Urinary Proteins (MUPs) are a class of lipocalins found in the urine of adult male mice which concentrate in the urine odorants which confer a characteristic odor. The behavioural as well as the endocrine effects of mouse urine and MUPs are briefly reviewed, suggesting a complex role is pheromonal communication. Some recent data on the molecular receptors of the vomeronasal organ further suggest a complex interaction with the MUP system.     

Immunoglobulin fold and tandem repeat structures in proteoglycan N-terminal domains and link protein

Detailed primary sequence and secondary structure analyses are reported for the hyaluronate binding region (G1 domain) and link protein of proteoglycan aggregates. These are based on six full or partial sequences from the chicken, pig, human, rat and bovine proteins. Determinations of a full pig and a partial human link protein sequence are reported in the Appendix. Five sequences at the N terminus in both proteins were compared with the structures of 11 variable immunoglobulin (Ig) fold domains for which crystal structures are available. Despite only modest sequence homology, a clear alignment could be proposed. Analysis of this shows that the equivalents of the first and second hypervariable segments are now significantly longer, and both proteins have N-terminal extensions that are up to 23 residues in length. Secondary structure predictions showed that these sequences could be identified with available crystal structures for the variable Ig fold. However the hydrophobic residues involved in interactions between the light and heavy chains in Igs are replaced by hydrophilic charged groups in both proteins. These results imply that both proteins are members of the Ig superfamily, but exhibit structural differences distinct from other members of this superfamily for which crystal structures are known. The proteoglycan tandem repeat (PTR) is a repeat of 99 residues that is found twice in the amino acid sequence of link protein and the proteoglycan G1 domain adjacent to the Ig fold, and also twice in the proteoglycan G2 domain. A total of 16 PTRs was available for analysis. Compositional analyses show that these are positively charged if these originate from link protein, and negatively charged if from the G1 or G2 domains. The 16 Robson secondary structure predictions for the PTRs were averaged to improve the statistics of the prediction, and checked by comparison with Chou-Fasman calculations. A strong alpha-helix prediction was found at residues 13 to 25, and several beta-strands were predicted. The overall content is 18% alpha-helix and 28% beta-sheet, with 44% of the remaining sequence being predicted as turns. These analyses show that both the proteoglycan G1 domain and link protein are constructed from two distinct globular components, which may provide the two functional roles of these proteins in proteoglycan aggregation.