Ideal architecture of residue packing and its observation in protein structures.

A simple model of sphere packing has been investigated as an ideal model for long-range interactions for the packing of non-bonded residues in protein structures. By superposing all residues, the geometry of packing around a central residue is investigated. It is found that all residues conform almost perfectly to this lattice model for sphere packing when a radius of 6.5 A is used to define non-bonded (virtual) interacting residues. Side-chain positions with respect to sequential backbone segments are relatively regular as well. This lattice can readily be used in conformation simulations to reduce the conformational space.     

Probing weakly polar interactions in cytochrome c.

Theoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and properly oriented aromatic rings. The two sulfur-containing amino acids, methionine and cysteine, occur frequently among functional alleles in random mutant libraries of Saccharomyces cerevisiae iso-1-cytochrome c genes at positions that form a weakly polar aromatic-aromatic interaction, the wild-type protein. To determine if a weakly polar sulfur-aromatic interaction replaced the aromatic-aromatic interaction, the structure and stability of two variants were examined. Phenylalanine 10, which interacts with tyrosine 97, was replaced by methionine and cysteine. The cysteine was modified to form the methionine and cysteine analog, S-methyl cysteine (CysSMe). Proton NMR studies indicate that changing Phe 10 to Met or CysSMe affects only local structure and that the structures of sulfur-containing variants are nearly identical. Analysis of chemical shifts and nuclear Overhauser effect data indicates that both sulfur-containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10CSMe variants are 2-3 kcal mol-1 less stable than iso-1-cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10CSMe variants allows evaluation of the potential weakly polar interaction between the additional sulfur atom of F10CSMe and the aromatic moiety of Tyr 97. The F10CSMe;C102T variant is 0.7 +/- 0.3 kcal mol-1 more stable than the F10M;C102T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur-containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aromatic ring is too weak to detect or is masked by destabilizing contributions to the free energy of denaturation.     

Cross-strand side-chain interactions versus turn conformation in beta-hairpins.

A series of designed peptides has been analyzed by 1H-NMR spectroscopy in order to investigate the influence of cross-strand side-chain interactions in beta-hairpin formation. The peptides differ in the N-terminal residues of a previously designed linear decapeptide that folds in aqueous solution into two interconverting beta-hairpin conformations, one with a type I turn (beta-hairpin 4:4) and the other with a type I + G1 beta-bulge turn (beta-hairpin 3:5). Analysis of the conformational behavior of the peptides studied here demonstrates three favorable and two unfavorable cross-strand side-chain interactions for beta-hairpin formation. These results are in agreement with statistical data on side-chain interactions in protein beta-sheets. All the peptides in this study form significant populations of the beta-hairpin 3:5, but only some of them also adopt the beta-hairpin 4:4. The formation of beta-hairpin 4:4 requires the presence of at least two favorable cross-strand interactions, whereas beta-hairpin 3:5 seems to be less susceptible to side-chain interactions. A protein database analysis of beta-hairpins 3:5 and beta-hairpins 4:4 indicates that the former occur more frequently than the latter. In both peptides and proteins, beta-hairpins 3:5 have a larger right-handed twist than beta-hairpins 4:4, so that a factor contributing to the higher stability of beta-hairpin 3:5 relative to beta-hairpin 4:4 is due to an appropriate backbone conformation of the type I + G1 beta-bulge turn toward the right-handed twist usually observed in protein beta-sheets. In contrast, as suggested previously, backbone geometry of the type I turn is not adequate for the right-handed twist. Because analysis of buried hydrophobic surface areas on protein beta-hairpins reveals that beta-hairpins 3:5 bury more hydrophobic surface area than beta-hairpins 4:4, we suggest that the right-handed twist observed in beta-hairpin 3:5 allows a better packing of side chains and that this may also contribute to its higher intrinsic stability.     

Crystal structures of the peanut lectin-lactose complex at acidic pH: retention of unusual quaternary structure, empty and carbohydrate bound combining sites, molecular mimicry and crystal packing directed by interactions at the combining site

The crystal structures of a monoclinic and a triclinic form of the peanut lectin-lactose complex, grown at pH 4.6, have been determined. They contain two and one crystallographically independent tetramers, respectively. The unusual "open" quaternary structure of the lectin, observed in the orthorhombic complex grown in neutral pH, is retained at the acidic pH. The sugar molecule is bound to three of the eight subunits in the monoclinic crystals, whereas the combining sites in four are empty. The lectin-sugar interactions are almost the same at neutral and acidic pH. A comparison of the sugar-bound and free subunits indicates that the geometry of the combining site is relatively unaffected by ligand binding. The combining site of the eighth subunit in the monoclinic crystals is bound to a peptide stretch in a loop from a neighboring molecule. The same interaction exists in two subunits of the triclinic crystals, whereas density corresponding to sugar exists in the combining sites of the other two subunits. Solution studies show that oligopeptides with sequences corresponding to that in the loop bind to the lectin at acidic pH, but only with reduced affinity at neutral pH. The reverse is the case with the binding of lactose to the lectin. A comparison of the neutral and acidic pH crystal structures indicates that the molecular packing in the latter is directed to a substantial extent by the increased affinity of the peptide loop to the combining site at acidic pH.     

Energetics of the lattice: Packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice—these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine · cytosine⁺ (C · C⁺) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other—for example, when folded in more complicated higher order structures, such as mRNA and chromatin. © 1998 John Wiley & Sons, Inc. Biopoly 44: 257–267, 1997     

Packing forces in nine crystal forms of cutinase

During the characterization of mutants and covalently inhibited complexes of Fusarium solani cutinase, nine different crystal forms have been obtained so far. Protein mutants with a different surface charge distribution form new intermolecular salt bridges or long-range electrostatic interactions that are accompanied by a change in the crystal packing. The whole protein surface is involved in the packing contacts and the hydrophobicities of the protein surfaces in mutual contact turned out to be noncorrelated, which indicates that the packing interactions are nonspecific. In the case of the hydrophobic variants, the packing contacts showed some specificity, as the protein in the crystal tends to form either crystallographic or noncrystallographic dimers, which shield the hydrophobic surface from the solvent. The likelihood of surface atoms to be involved in a crystal contact is the same for both polar and nonpolar atoms. However, when taking areas in the 200–600 Ų range, instead of individual atoms, the either highly hydrophobic or highly polar surface regions were found to have an increased probability of establishing crystal lattice contacts. The protein surface surrounding the active-site crevice of cutinase constitutes a large hydrophobic area that is involved in packing contacts in all the various crystalline contexts. Proteins 31:320–333, 1998. © 1998 Wiley-Liss, Inc.     

Intra- versus intermolecular interactions in monellin: contribution of surface charges to protein assembly

The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments. The reconstitution of monellin mutants containing large surface charge perturbations was compared to the thermostability of structurally equivalent single-chain monellin containing the same sets of mutations under varying salt concentrations. The affinity between monellin fragments is found to correlate with the thermostability of single chain monellin, indicating the involvement of the same underlying Coulombic interactions. This confirms that there are no principal differences in the interactions involved in folding and binding. Based on comparison with a previous mutational study involving hydrophobic core residues, the relative contribution of Coulombic interactions to stability and affinity is modest. However, the Coulombic perturbations only affect the association rates of reconstitution in contrast to perturbations involving hydrophobic residues, which affect primarily the dissociation rates. These results indicate that Coulombic interactions are likely to be of main importance for the association of protein assembly, relevant for functions of proteins.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

MD simulation of subtilisin BPN' in a crystal environment.

In this paper we present a molecular dynamics (MD) simulation of subtilisin BPN' in a crystalline environment containing four protein molecules and solvent. Conformational and dynamic properties of the molecules are compared with each other and with respect to the X-ray structure to test the validity of the force field. The agreement between simulated and experimental structure using the GROMOS force field is better than that obtained in the literature using other force fields for protein crystals. The overall shape of the molecule is well preserved, as is the conformation of alpha-helices and beta-strands. Structural differences are mainly found in loop regions. Solvent networks found in the X-ray structure were reproduced by the simulation, which was unbiased with respect to the crystalline hydration structure. These networks seem to play an important role in the stability of the protein; evidence of this is found in the structure of the active site. The weak ion binding site in the X-ray structure of subtilisin BPN' is occupied by a monovalent ion. When a calcium ion is placed in the initial structure, three peptide ligands are replaced by 5 water ligands, whereas a potassium ion retains (in part) its original ligands. Existing force fields yield a reliable method to probe local structure and short-time dynamics of proteins, providing an accuracy of about 0.1 nm.     

Crystal structures of the two membrane-proximal Ig-like domains (D3D4) of LILRB1/B2: alternative models for their involvement in peptide-HLA binding

Leukocyte immunoglobulin-like receptors (LILRs), also called CD85s, ILTs, or LIRs, are important mediators of immune activation and tolerance that contain tandem immunoglobulin (Ig)-like folds. There are 11 (in addition to two pseudogenes) LILRs in total, two with two Ig-like domains (D1D2) and the remaining nine with four Ig-like domains (D1D2D3D4). Thus far, the structural features of the D1D2 domains of LILR proteins are well defi ned, but no structures for the D3D4 domains have been reported. This is a very important fi eld to be studied as it relates to the unknown functions of the D3D4 domains, as well as their relative orientation to the D1D2 domains on the cell surface. Here, we report the crystal structures of the D3D4 domains of both LILRB1 and LILRB2. The two Iglike domains of both LILRB1-D3D4 and LILRB2-D3D4 are arranged at an acute angle (~60°) to form a bent structure, resembling the structures of natural killer inhibitory receptors. Based on these two D3D4 domain structures and previously reported D1D2/HLA I complex structures, two alternative models of full-length (four Ig-like domains) LILR molecules bound to HLA I are proposed.     

Defining oligosaccharide specificity for initial sperm-zona pellucida adhesion in the mouse

The identity of the sperm surface protein(s) responsible for sperm-zona pellucida binding in the mouse, as well as the characteristics of the oligosaccharide groups on zona pellucida glycoprotein 3 (ZP3) having ligand activity toward this receptor, remain controversial. Conflicting results from several groups have made interpretation of the current data difficult. By developing a quantitative binding assay to evaluate the molecular interactions between mammalian sperm and the zona pellucida during initial gamete interactions, we directly quantified sperm-ZP binding interactions at the molecular level for the first time. The ZP binding assay demonstrated that live, capacitated mouse sperm bind solubilized ¹²⁵I-labeled ZP glycoproteins in a concentration-dependent manner characterized by a rapid forward rate constant of 3.0 × 10⁷ M⁻¹ min⁻¹. Following the initial characterization, the binding assay was used to examine the roles of the sperm surface enzymes galactosyltransferase (GalTase) and fucosyltransferase (FucTase) in sperm-zone pellucida binding in the mouse. These data indicate that substrates for FucTase, but not for GalTase, inhibit sperm-ZP binding, in contrast to earlier reports in which GalTase substrates significantly inhibited sperm binding to intact ZPs. A model is presented which resolves conflicting results between assays using intact ZPs and the results obtained here using soluble ¹²⁵I-ZPs. Assuming a complex binding/recognition site, monosaccharides that could occupy part of the binding site would have a dramatic effect on sperm-ZP binding to the intact ZP, since they need only occupy the binding sites for a short time (∼ 100 msec) to disrupt binding. The current results suggest that the sperm ZP3 receptor binding site minimally recognizes the galβ1,3GlcNAc moiety also recognized by FucTases. The current data do not exclude the possibility that additional sugar residues form part of the ligand oligosaccharide group and are recognized by a yet-to-be-identified sperm surface protein which serves as the ZP3 receptor. © 1996 Wiley-Liss, Inc.     

Tests for helix-stabilizing interactions between various nonpolar side chains in alanine-based peptides.

Straight-chain, non-natural, nonpolar amino acids norleucine, norvaline, and alpha-amino-n-butyric acid at various spacings do not interact with themselves to stabilize helix formation in alanine-based peptides, but do interact with a Tyr spaced i, i + 4 to stabilize alanine helices, similar to the helix-stabilizing i, i + 4 Tyr-Leu and Tyr-Val interactions reported earlier (Padmanabhan S, Baldwin RL, 1994, J Mol Biol 241:706-713). Leu spaced i, i + 4 from another Leu is measurably helix-stabilizing relative to the corresponding i, i + 3 pair, but less so than for i, i + 4 Val-Leu, Ile-Leu, or Phe-Leu pairs (relative to the corresponding i, i + 3 pairs) when Leu is C-terminal to the other nonpolar amino acid. Our results indicate that limited side-chain flexibility in an alpha-helix strongly favors the interaction between 2 nonpolar residues to stabilize an isolated alpha-helix.     

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer.     

Regional interactions of opioid peptides at μ and δ sites in rat brain

Opiate binding sites in five brain regions were labeled with the μ and δ markers, ³H-morphine and ³H-[D-Ala²,D-leu⁵]enkephalin, respectively. The highest densities of both ³H-morphine and ³H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly ³H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala²,D-leu⁵]enkephalin, β-endorphin and dynorphin(1–13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the μ and δ markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the δ binding site and β-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1–13) shows a high affinity and selective preference for the μ binding site over the δ site. The potency of the opioid peptides in displacing the μ and δ markers varies from region to region according to the relative densities of the two opiate binding site populations.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Protein-protein interactions: hot spots and structurally conserved residues often locate in complemented pockets that pre-organized in the unbound states: implications for docking

Energetic hot spots account for a significant portion of the total binding free energy and correlate with structurally conserved interface residues. Here, we map experimentally determined hot spots and structurally conserved residues to investigate their geometrical organization. Unfilled pockets are pockets that remain unfilled after protein-protein complexation, while complemented pockets are pockets that disappear upon binding, representing tightly fit regions. We find that structurally conserved residues and energetic hot spots are strongly favored to be located in complemented pockets, and are disfavored in unfilled pockets. For the three available protein-protein complexes with complemented pockets where both members of the complex were alanine-scanned, 62% of all hot spots (DeltaDeltaG>2kcal/mol) are within these pockets, and 60% of the residues in the complemented pockets are hot spots. 93% of all red-hot residues (DeltaDeltaG>/=4kcal/mol) either protrude into or are located in complemented pockets. The occurrence of hot spots and conserved residues in complemented pockets highlights the role of local tight packing in protein associations, and rationalizes their energetic contribution and conservation. Complemented pockets and their corresponding protruding residues emerge among the most important geometric features in protein-protein interactions. By screening the solvent, this organization shields backbone hydrogen bonds and charge-charge interactions. Complemented pockets often pre-exist binding. For 18 protein-protein complexes with complemented pockets whose unbound structures are available, in 16 the pockets are identified to pre-exist in the unbound structures. The root-mean-squared deviations of the atoms lining the pockets between the bound and unbound states is as small as 0.9A, suggesting that such pockets constitute features of the populated native state that may be used in docking.     

Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses.     

Fast and automated functional classification with MED‐SuMo: An application on purine‐binding proteins

Ligand–protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED‐SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED‐SMA is an automated and fast method to classify binding sites. It is based on MED‐SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora‐A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED‐SMA approach is demonstrated as it gathers binding sites of proteins with similar structure‐activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target‐based drug design and prediction of cross‐reactivity and therefore potential toxic side effects.     

Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site.

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide-linked to apolipoprotein B-100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2'-azobis-(amidinopropane) HCl (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r-apo(a), to bind through their LBS domains. AAPH treatment caused a time-dependent decrease in the number of functional Lp(a) or r-apo(a) molecules capable of binding to fibrin or lysine-Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r-apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle-containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r-apo(a) was observed as assessed by native or SDS-PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl-containing compounds increase the LBS affinity of Lp(a) for fibrin.