Cooligopeptides containing aromatic residues spaced by glycyl residues. IX. Fluorescence properties of tryptophan-containing peptides

The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λ_max) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)_n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λ_max, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λ_max of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. II. Ultraviolet absorption and circular dichroism properties of co-oligopeptides of tryptophan and glycine

The uv absorption and circular dichroism (CD) properties in water (pH 5.9) and trifluoroethanol of several co-oligopeptides of glycine and tryptophan have been investigated. These compounds contain one tryptophyl residue, such as H-Gly-Trp-OH, H-Trp-Gly-OH, and H-Gly-Trp-Gly-OH; or two, such as H-Trp-Trp-OH, H-Gly-Trp-Trp-OH, H-Trp-Trp-Gly-OH, and H-Gly-Trp-(Gly)_n-Trp-Gly-OH (I, n = 0,1,2). Furthermore, the case of some protected derivatives, such as Z-Trp-Gly-OMe, Z-Gly-Trp-Gly-OMe, and Z-Trp-Trp-OMe, Z-Trp-Trp-Gly-OMe, Z-Gly-Trp-Trp-Ome were investigated. The extinction coefficients in H₂O in the region of the L bands, referred to one mole of tryptophyl residue, are essentially the same for all compounds within the limit of experimental error, thus indicating that no strong chromophoric interaction takes place in the oligopeptides containing two aromatic residues. However, in the far uv region, anomalies in the absorption properties cannot be excluded. The investigated compounds show marked differences in their CD properties. Whereas in the case of lower molecular-weight peptides (e.g., H-Trp-Gly-OH and H-Gly-Trp-OH), these differences can be ascribed, at least in part, to the influence of the charge end groups on the indole chromophore, in the case of the compounds containing two tryptophyl residues, the differences in CD properties are assumed to reflect the different structures and conformations of the oligopeptides. In particular, we observed a negative dichroic band at ～225 nm in H-Gly-Trp-Trp-OH and I(n = 0) in water. In trifluoroethanol, this band has a much larger intensity, and it is present also in Z-Gly-Trp-Trp-OMe, Z-Trp-Trp-Gly-OMe, and Z-Trp-Trp-OMe. It is argued that such a negative band is characteristic of the sequence -CO-Trp-Trp-, and the possibility that it derives from intramolecular chromophoric interaction is discussed. It is also pointed out, particularly in view of the high ellipticity values found in some cases (up to 100,000 deg cm² dmol^?1), that this feature may reflect a high degree of conformational rigidity connected with the sequence -CO-Trp-Trp-.     

Macromolecular properties of cepacian in water and in dimethylsulfoxide

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.     

A Study of stepwise conformational changes in poly(β-benzyl-L-aspartate) with the helix–coil transition by means of proton and carbon-13 NMR

The helix–coil transition for poly(β-benzyl-L -aspartate) [poly(Asp[OBzl])] in solvent mixtures of trifluoroacetic acid/deuterated chloroform (F₃AcOH/CDCl₃) was studied by means of proton and carbon-13 nmr. Conformational fixation of the side chain occurs before the coil–helix transition of the backbone, when neighboring phenyl rings face each other. Another type of conformational fixation occurs in the side chain after the coil–helix transition of the backbone. These conformational changes of the side chain are due to the changes of the strength of the interaction between the side-chain ester group and the F₃AcOH molecule. In the absence of F₃AcOH (coil-forming solvent), the polymer has a rather rigid structure in which the side chain may wrap around the backbone. These conformational changes of the polymer are closely related to the changes of the interaction between the polymer and F₃AcOH molecules.     

Mechanistic aspects of chiral discrimination by surface‐immobilized α1‐acid glycoprotein

The immobilization of the globular protein α‐1‐acid glycoprotein (AGP) onto silica gel led to the commercial availability of an AGP column, which has a high enantioselectivity. The enantioselectivity of AGP columns has been demonstrated in numerous applications. Due to potential AGP structural changes occurring upon its immobilization, the interaction between particular pairs of enantiomers and the stationary phase is very difficult to assess. Therefore, in this paper we report a mechanistic study that probes the nature of these types of interactions. As model ligands, we employed two LTD₄ antagonists (L‐708, 738, MK0476, and their enantiomers) which have a rigid backbone consisting of a conjugated aromatic region and a side chain which is terminated with a carboxylic functional group. The difference between the two compounds is a two‐fluorine versus one‐chlorine substituent in the aromatic region of the molecule. To study the interaction between the two homologues and the AGP stationary phase, several parameters were varied, including pH, ionic strength, organic modifier, and temperature. van't Hoff plots were constructed and found to be nonlinear. They could, however, be divided into two linear regions, one from 0°C to ∼30°C, and another from 39°C to 50°C. The region at lower temperature implied that the separation was entropy‐dominated while the separation at higher temperature was enthalpically driven. The transition from the entropic to the enthalpically driven separation region suggested that bound AGP undergoes a conformational change. Fluorescence spectroscopy performed on the AGP stationary phase found evidence for a limited conformational transition at a similar temperature, consistent with this hypothesis. Chirality 11:224–232, 1999. © 1999 Wiley‐Liss, Inc.     

Co-oligopeptides of aromatic amino acids and glycine with variable distance between the aromatic residues. VII. Enzymatic synthesis of N-protected peptide amides

Several N-protected peptide amides, containing two aromatic residues spaced by one glycyl residue, have been enzymatically synthesized starting from P-Ar-OH and H-Gly-Ar-NH₂ (P is the protecting group and Ar is the aromatic residue) and using α-chymotrypsin as the catalyst for the coupling step. Reactions have been carried out in water solution, at room temperature, and afford yields ranging between 20 and 75% ca. This coupling reaction occurs in a much more restricted set of conditions than the hydrolysis reaction, e.g., only within a small pH range (ca. 6.5–7.5) and with particular buffering agents. The advantages and limitations of this type of reaction, compared with conventional coupling procedures, are discussed.     

δ-Selective Opioid Peptides Containing a Single Aromatic Residue in the Message Domain: An NMR Conformational Analysis

The sequence of deltorphin I, a δ-selective opioid agonist, has been systematically modified by inserting conformationally constrained C^α,α disubstituted apolar residues in the third position. As expected, substitution of Phe with Ac₆c, Ac₅c and Ac₃c yields analogues with decreasing but sizeable affinity. Surprisingly, substitution with Aib yields an analogue with almost the same binding affinity of the parent compound but with a greatly increased selectivity. This is the first case of a potent and very selective opioid peptide containing a single aromatic residue in the message domain, that is, only Tyr¹. Here we report a detailed conformational analysis of [Aib³]deltorphin I and [Ac₆c³]deltorphin I in DMSO at room temperature and in a DMSO/water cryomixture at low temperature, based on NMR spectroscopy and energy calculations. The peptides are highly structured in both solvents, as indicated by the exceptional finding of a nearly zero temperature coefficient of Val⁵ NH resonance. NMR data cannot be explained on the basis of a single structure but it was possible to interpret all NMR data on the basis of a few structural families. The conformational averaging was analysed by means of an original computer program that yields qualitative and quantitative composition of the mixture. Comparison of the preferred solution conformations with two rigid δ-selective agonists shows that the shapes of [Aib³]deltorphin I and [Ac₆c³]deltorphin I are consistent with those of rigid agonists and that the message domain of opioid peptides can be defined only in conformational terms.     

Molecular orbital calculations on the conformation of polypeptides and proteins. II. Conformational energy maps and stereochemical rotational states of aromatic residues

Our previous quantum-mechanical calculations, by an all-valence-electrons method (PCILO) taking into account simultaneously the σ and π electrons of the system, on the conformation energy maps of the glycyl and alanyl residues are extended to the evaluation of these maps and of the stereochemical rotational states of the aromatic residues, phenylalanyl, tyrosyl, histidyl, and tryptophanyl in dipeptides. Calculations on model compounds are used for the predetermination of the side-chain rotational angles χ₁ and χ₂ which are then used as selected parameters for the evaluation of the conformational energy maps as function of the backbone rotational angles Φ and ψ. The theory predicts that the most stable conformation for these aromatic residues should occur in the same region, around Φ = 200, ψ = 140°, in which it was predicted to occur for the glycyl and alanyl residues and which was completely overlooked by most of the previous “empirical” computations. Recent experimental work by a group of Russian authors using NMR and infrared techniques seems to confirm the theoretical result for the alanyl and phenylalanyl residues. The paper indicates also the secondary local minima which appear for the different residues. The theoretically allowed general conformational area for the four aromatic residues, within the reasonable value of 5 kcal/mole above the deepest minimum, is somewhat larger than the similar area allowed by the “hard sphere” empirical calculations. Practically all available representative experimental points from the study of small molecules and of the proteins lysozyme and myoglobin fall within the allowed area, the agreement being better with the results of the quantum mechanical calculations than with those of the “hard sphere” approximation. The values of the side-chain rotational angles χ₁ and χ₂ and of their allowed combinations agree less satisfactorily with experiment, the experimentally observed combinations being more varied than the theoretically allowed ones. These last ones having, however, been predetermined on studies with model compounds, this situation is not astonishing. It is proposed to refine these results by a minimization with respect to the four parameters Φ, ψ, χ₁, and χ₂ involved.     

Interactions of aromatic residues of proteins with nucleic acids. Fluorescence studies of the binding of oligopeptides containing tryptophan and tyrosine residues to polynucleotides.

The binding of oligopeptides of general structure Lys-X-Lys (where X is an aromatic residue) to several polynucleotides has been studied by fluorescence spectroscopy. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups as shown by the ionic strength and pH dependence of binding. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the nucleic acid bases with the aromatic amino acid whose fluorescence is quenched. Fluorescence data have been quantitatively analyzed according to a model involving these two types of complexes. Association constants and the size of binding sites have been determined. Stacking interactions are favored in single-stranded polynucleotides as compared to double-stranded ones. A short oligopeptide such as Lys-X-Lys is thus able to distinguish between single-stranded and double-stranded nucleic acids. Fluorescence results are compared to those obtained by proton magnetic resonance and circular dichroism.     

Analysis of the aromatic 1H-NMR spectrum of the kringle 5 domain from human plasminogen. Evidence for a conserved kringle fold

A kringle 5 domain fragment from human plasminogen has been investigated by 1H-NMR spectroscopy at 300 MHz and 620 MHz. The study focuses on the kringle 5 aromatic spectrum as aromatic side chains appear to mediate the binding of benzamidine. Spin-echo experiments and acid/base-titration studies in conjunction with two-dimensional double-quantum and chemical-shift-correlated spectroscopies were used to identify individual spin systems. Sequence-specific assignments of aromatic resonances are derived from direct comparison of the kringle 5 spectrum with spectra of the homologous kringle 1 and kringle 4 domains of plasminogen. As previously observed for kringles 1 and 4, the pattern we detect for Tyr9 in kringle 5 reflects a slow conformational exchange between two states in equilibrium, one in which the Tyr9 ring is freely mobile and one in which its flip dynamics are constrained. Proton Overhauser experiments in 1H2O and in 2H2O have been used to probe aromatic ring interactions and to identify residues which are part of the hydrophobic core centered at the Leu46 side chain. Overall, the data indicate a strong structural homology among the three plasminogen kringles.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. VI. Circular dichroism studies of Co-oligopeptides of l-phenylalanine, L-tryptophan, and glycine.

The circular dichroic properties of H-Gly-Phe-(Gly)_n-Trp-Gly-OH (II, n = 0,1,2) and of related simpler peptides, such as H-Phe-Gly-OH, H-Gly-Phe-OH, H-Gly-Phe-Gly-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, and H-Gly-Phe-Trp-OH in water and trifluoroethanol solutions are investigated. Peptides containing only one phenylalanyl residue show markedly different near-uv dichroic signals depending on whether this residue is in the N-terminal position or not. The possible origin of this feature is discussed. The study of the oligopeptides II (n = 0,1,2) shows that no strong intramolecular interaction between the two aromatic rings is present. However, the dichroic properties of II (n = 0) are clearly anomalous, and the analysis of H-Gly-Phe-Trp-OH, H-Phe-Trp-Gly-OH, and of H-Phe-Trp-OH at different pH's, confirms that the presence of two adjacent aromatic residues may bring about chiroptical properties which indicate a restriction in the conformational equilibrium of the molecule. The limits, and the possible generalization of this finding, are discussed.     

A Comparative Molecular Dynamics Study of Thermophilic and Mesophilic Ribonuclease HI Enzymes

Abstract

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.     

How different are structurally flexible and rigid binding sites? Sequence and structural features discriminating proteins that do and do not undergo conformational change upon ligand binding

Proteins are dynamic molecules and often undergo conformational change upon ligand binding. It is widely accepted that flexible loop regions have a critical functional role in enzymes. Lack of consideration of binding site flexibility has led to failures in predicting protein functions and in successfully docking ligands with protein receptors. Here we address the question: which sequence and structural features distinguish the structurally flexible and rigid binding sites? We analyze high-resolution crystal structures of ligand bound (holo) and free (apo) forms of 41 proteins where no conformational change takes place upon ligand binding, 35 examples with moderate conformational change, and 22 cases where a large conformational change has been observed. We find that the number of residue-residue contacts observed per-residue (contact density) does not distinguish flexible and rigid binding sites, suggesting a role for specific interactions and amino acids in modulating the conformational changes. Examination of hydrogen bonding and hydrophobic interactions reveals that cases that do not undergo conformational change have high polar interactions constituting the binding pockets. Intriguingly, the large, aromatic amino acid tryptophan has a high propensity to occur at the binding sites of examples where a large conformational change has been noted. Further, in large conformational change examples, hydrophobic-hydrophobic, aromatic-aromatic, and hydrophobic-polar residue pair interactions are dominant. Further analysis of the Ramachandran dihedral angles (phi, psi) reveals that the residues adopting disallowed conformations are found in both rigid and flexible cases. More importantly, the binding site residues adopting disallowed conformations clustered narrowly into two specific regions of the L-Ala Ramachandran map. Examination of the dihedral angles changes upon ligand binding shows that the magnitude of phi, psi changes are in general minimal, although some large changes particularly between right-handed alpha-helical and extended conformations are seen. Our work further provides an account of conformational changes in the dihedral angles space. The findings reported here are expected to assist in providing a framework for predicting protein-ligand complexes and for template-based prediction of protein function.     

Peptide hairpins with strand segments containing alpha- and beta-amino acid residues: cross-strand aromatic interactions of facing Phe residues

The incporation of beta-amino acid residues into the strand segments of designed beta-hairpin leads to the formation of polar sheets, since in the case of beta-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of alpha/beta-hybrid segments are described: Boc-Leu-betaPhe-Val-(D)-Pro-Gly-Leu-betaPhe-Val-OMe (1) and Boc-betaLeu-Phe-betaVal-D-Pro-Gly-betaLeu-Phe-betaVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a beta-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II' beta-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the betaPhe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the beta-sheet. The observed aromatic centroid-to-centroid distances are 8.92 A (molecule A) and 8.94 A (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure.Peptide 1 yields a normal "hairpin-like" CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical beta-hairpin suggests that interring separation is approximately 4.78 A for the gauche+ gauche- (g+ g-) rotamer. A previously reported peptide beta-hairpin composed of only alpha-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also exhibited an anomalous far-UV (ultraviolet) CD (circular dichroism) spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (C. Zhao, P. L. Polavarapu, C. Das, and P. Balaram, Journal of the American Chemical Society, 2000, Vol 122, pp. 8228-8231).     

In silico analysis of conformational changes induced by mutation of aromatic binding residues: consequences for drug binding in the hERG K+ channel

Pharmacological inhibition of cardiac hERG K(+) channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences.     

Influence of hydrogen bonding,main chain–side chain interactions,and protecting groups on the excimer formation of bis(pyrenylalanine) peptides

Dipeptides of the aromatic fluorescent amino acid, pyrenylalanine, are studied using both stationary and transient fluorescence techniques. Since the conformational transitions of the peptide chain are slow compared to the decay of the pyrene excited state, both ground state conformations, adopted by the peptide, i.e., C₅ and C₇, can be monitored separately. Kinetic models are proposed to describe the molecular dynamics of the peptide chain as probed by the intramolecular excimer formation between both pyrene chromophores. These kinetic schemes explain the influence of solvent, chain chirality, main chain–side chain interactions, and nature of the protecting groups on the emission spectrum and the fluorescence decay profile of these model peptides. These schemes also provide a tool to calculate rate constants of conformational transitions and excimer formation. By comparing the kinetic and thermodynamic parameters of the various compounds, the influence of a structural modification on the molecular dynamics of the peptide chain is determined.     

Exciton circular dichroism couplet arising from nitrile-derivatized aromatic residues as a structural probe of proteins

Exciton coupling between two chromophores can produce a circular dichroism (CD) couplet that depends on their separation distance, among other factors. Therefore, exciton CD signals arising from aromatic sidechains, especially those of tryptophan (Trp), have been used in various protein conformational studies. However, the long-wavelength component of the commonly used CD couplet produced by a pair of Trp residues is typically located around 230 nm, thereby overlapping significantly with the protein backbone CD signal. This overlap often prevents a direct and quantitative assessment of the Trp CD couplet in question without further spectral analysis. Here, we show that this inconvenience can be alleviated by using a derivative of Trp, 5-cyanotryptophan (Trp_CN), as the chromophore. Specifically, through studying a series of peptides that fold into either α-helical or ß-hairpin conformations, we demonstrate that in comparison with the Trp CD couplet, that arising from two Trp_CN residues not only is significantly red-shifted but also becomes more intense due to the larger extinction coefficient of the underlying electronic transition. In addition, we show that a pair of p-cyanophenylalanines (Phe_CN) or a Phe_CN–Trp_CN pair can also produce a distinct exciton CD couplet that can be useful in monitoring conformational changes in proteins.     

A comparative molecular dynamics study of thermophilic and mesophilic ribonuclease HI enzymes

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

Correlation between the exposure of aromatic chromophores at the surface of the Fc domains of immunoglobulin G and their ability to bind complement.

The recognition that certain biological effector functions associated with the Fc region of human IgG are mediated exclusively by either the Cgamma2 or Cgamma3 domains prompted a study of some of the physical properties of the isolated domains in an attempt to correlate these with functional differentiation. The degree of aromatic chromophore exposure of intact Fc and fragments corresponding to the Cgamma2 and Cgamma3 domains were determined by solvent perturbation difference spectroscopy using 20% ethylene glycol. For the monomeric Cgamma2 fragment one of the two tryptophans and all four of the tyrosines were exposed to solvent. In the pFc' fragment, which represented a dimer of two intact Cgamma3 domains, an average of 0.4 of the two tryptophans of 3.3 of the five tyrosines per chain were exposed. These data were consistent with the suggested involvement of tryptophan in complement fixation since Cgamma2 binds C1q but pFc' does not. Several fragments derived from the Cgamma3 region had previously been shown to have differing environments for their aromatic side chains from circular dichroism studies. These fragments have now been shown to exhibit different degrees of chromophore exposure to solvent. Removal of the carboxy-termimal heptapeptide from the intact, Cgamma3 domain resulted in a fragment not only showing a greater exposure of aromatic residues but also having the ability to bind Clq. Our data suggest that the structural requirements for C1Q binding may be quite commonplace within Fc, but tertiary folding limits their expression except in Cgamma2 in the native molecule. The solvent perturbation observed with Fc was somewhat lower than would have been expected from the results with the isolated domains, suggesting that interdomain interactions may result in burial of aromatic residues.