Short leucine-rich glycoproteins of the extracellular matrix display diverse patterns of complement interaction and activation.

The extracellular matrix consists of structural macromolecules and other proteins with regulatory functions. An important family of the latter class of molecules found in most tissues is the small leucine-rich repeat proteins (SLRPs). We have previously shown that the SLRP fibromodulin binds directly to C1q and activates the classical pathway of complement. In the present study we further examine the interactions between SLRPs and complement. Osteoadherin, like fibromodulin, binds C1q and activates the classical pathway strongly while moderate activation is seen in the terminal pathway. This can be explained by the interaction of fibromodulin and osteoadherin with factor H, a major soluble inhibitor of complement. Also, chondroadherin was found to bind C1q and activate complement, albeit to a lesser extent. Chondroadherin also binds factor H. We confirm published data showing that biglycan and decorin bind C1q but do not activate complement. In this study a similar pattern is seen for lumican although its affinity for C1q is lower than for biglycan and decorin. Furthermore, using electron microscopy and radiolabeled SLRPs, we demonstrate two different classes of SLRP binding sites on C1q, to head and stalk respectively, where only binding to the head appears to be activating. We propose a role for SLRPs in the regulation of complement activation in diseases involving the extracellular matrix, particularly those characterized by chronic inflammation such as rheumatoid arthritis, atherosclerosis, osteoarthritis and chronic obstructive lung disease.

Binding of the volatile general anesthetics halothane and isoflurane to a mammalian beta-barrel protein.

A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic-protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large beta-barrel internal cavity (515 +/- 30 angstroms(3)) lined predominantly by aromatic and aliphatic residues. Halothane binding to the beta-barrel cavity was determined using fluorescence quenching of Trp16, and a competitive binding assay with 1-aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using isothermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the beta-barrel of porcine odorant binding protein with dissociation constants of 0.46 +/- 0.10 mM and 0.43 +/- 0.12 mM determined using fluorescence quenching and competitive binding with 1-aminoanthracene, respectively. Isothermal titration calorimetry revealed that halothane and isoflurane bound with K(d) values of 80 +/- 10 microM and 100 +/- 10 microM, respectively. Halothane and isoflurane binding resulted in an overall stabilization of the folded conformation of the protein by -0.9 +/- 0.1 kcal.mol(-1). In addition to indicating specific binding to the native protein conformation, such stabilization may represent a fundamental mechanism whereby anesthetics reversibly alter protein function. Because porcine odorant binding protein has been successfully analyzed by X-ray diffraction to 2.25 angstroms resolution [1], this represents an attractive system for atomic-level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules.

Towards a three-alpha-helix bundle protein that binds volatile general anesthetics.

The general anesthetics halothane and chloroform are capable of binding to synthetic water-soluble four-alpha-helix bundles, which model the putative in vivo receptors. In this study, we investigate the binding of these anesthetics to synthetic water-soluble three-alpha-helix bundles. A series of variants containing up to four X-to-Ala and up to four X-to-Met substitutions was made; and the effect of these substitutions on structure, stability and anesthetic binding affinity was examined. Generally, the amount of alpha-helix and the stability of the three-alpha-helix bundles decreased as the number of X-to-Ala substitutions increased. A concomitant red-shift in tryptophan fluorescence lambdamax was seen, suggesting an increased flexibility of the native structure. Up to four X-to-Met substitutions had little effect on the amount of alpha-helix, but an increase in tryptophan lambdamax was seen for the variants with three and four methionine substitutions. The exceptions were a) a variant with a clustering of alanine and methionine residues at one end of the three-alpha-helix bundle, suggesting a gate structure that can admit ligand molecules; and b) a variant with a single Leu35Ala substitution, suggesting that at select positions, the size of the side chain is important for defining anesthetic binding affinity.

Redesigning the folding energetics of a model three-helix bundle protein by site-directed mutagenesis.

Because of their limited size and complexity, de novo designed proteins are particularly useful for the detailed investigation of folding thermodynamics and mechanisms. Here, we describe how subtle changes in the hydrophobic core of a model three-helix bundle protein (GM-0) alter its folding energetics. To explore the folding tolerance of GM-0 toward amino acid sequence variability, two mutant proteins (GM-1 and GM-2) were generated. In the mutants, cavities were created in the hydrophobic core of the protein by either singly (GM-1; L35A variant) or doubly (GM-2; L35A/I39A variant) replacing large hydrophobic side chains by smaller Ala residues. The folding of GM-0 is characterized by two partially folded intermediate states exhibiting characteristics of molten globules, as evidenced by pressure-unfolding and pressure-assisted cold denaturation experiments. In contrast, the folding energetics of both mutants, GM-1 and GM-2, exhibit only one folding intermediate. Our results support the view that simple but biologically important folding motifs such as the three-helix bundle can reveal complex folding plasticity, and they point to the role of hydrophobic packing as a determinant of the overall stability and folding thermodynamic of the helix bundle.

Effect of four-alpha-helix bundle cavity size on volatile anesthetic binding energetics.

Currently, it is thought that inhalational anesthetics cause anesthesia by binding to ligand-gated ion channels. This is being investigated using four-alpha-helix bundles, small water-soluble analogues of the transmembrane domains of the "natural" receptor proteins. The study presented here specifically investigates how multiple alanine-to-valine substitutions (which each decrease the volume of the internal binding cavity by 38 A(3)) affect structure, stability, and anesthetic binding affinity of the four-alpha-helix bundles. Structure remains essentially unchanged when up to four alanine residues are changed to valine. However, stability increases as the number of these substitutions is increased. Anesthetic binding affinities are also affected. Halothane binds to the four-alpha-helix bundle variants with 0, 1, and 2 substitutions with equivalent affinities but binds to the variants with 3 and 4 more tightly. The same order of binding affinities was observed for chloroform, although for a particular variant, chloroform was bound less tightly. The observed differences in binding affinities may be explained in terms of a modulation of van der Waals and hydrophobic interactions between ligand and receptor. These, in turn, could result from increased four-alpha-helix bundle binding cavity hydrophobicity, a decrease in cavity size, or improved ligand/receptor shape complementarity.

Role of aromatic side chains in the binding of volatile general anesthetics to a four-alpha-helix bundle.

Currently, the mechanism by which anesthesia occurs is thought to involve the direct binding of inhaled anesthetics to ligand-gated ion channels. This hypothesis is being studied using four-alpha-helix bundles as model systems for the transmembrane domains of the natural "receptor" proteins. This study concerns the role in anesthetic binding played by aromatic side chains in the binding cavity of a four-alpha-helix bundle designed to assume a Rop-like fold. Specifically, the effect of the substitution W15Y on bundle structure, stability, and anesthetic binding energetics was investigated. No appreciable effect of substituting W for Y on the secondary structure or the thermodynamic stability of the four-alpha-helix bundle was identified. However, the substitution W15Y resulted in about 6- and 3-fold decreases in halothane and chloroform binding affinities, respectively. This effect may reflect weaker dipole-aromatic quadrupole interactions between the aromatic side chain and the anesthetic in the tyrosine-containing species, which possesses the smaller aromatic ring system. For these anesthetic binding proteins, this class of interaction occurs when the permanent nonspherical distribution of electrons in the aromatic ring systems interact with the weakly acidic CH group of the anesthetics.