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.

Rotamer-specific fluorescence quenching in tyrosinamide: Dynamic and static interactions.

We have examined the environments of the three phenol rotamers about the C(α)-C(ß) bond in tyrosinamide by fluorescence quenching. Steady-state acrylamide quenching yields a nonlinear stern-Volmer plot. With three distinct emitting species and no other information about the system, it is impossible to analyze the data due to the number of variables which have to be determined. We therefore reduced the number of variables by independently determining the fractional intensity and dynamic quenching constant for each rotamer through time-resolved fluorescence quenching studies. These parameters were then used to analyze the steady-state data for any contribution of static quenching. We conclude that the nonlinear Stern-Volmer plot for the quenching of tyrosinamide by acrylamide is a consequence of each rotamer having a distinct dynamic quenching constant and the presence of static quenching. The static quenching can be represented by either the sphere-of-action model involving two of the three rotamers or the ground-state complex model involving all three rotamers.

The mechanism of quenching of liver alcohol dehydrogenase fluorescence due to ternary complex formation.

Difference fluorescence emission spectra, reciprocal Stern-Volmer plots, and variable excitation wave-lengths have been used to evaluate the selective quenching of the two tryptophan residues/subunit of liver alcohol dehydrogenase. Trp-15, at the surface of the enzyme, is quenched by KI consistent with a collisional mechanism, and has a blue-shifted excitation and red-shifted emission spectrum when compared with the spectral properties of TRP-314, which is in a hydrophobic milieu at the subunit interface of the dimeric enzyme. With excitation at 295 nm, Trp-314 is 80% quenched by formation of a ternary enzyme.NAD+.trifluoroethanol complex, and the quenching is essentially additive to that caused by KI. Alkaline pH also results in selective quenching of Trp-314. These results, and considerations of the three-dimensional structure of the enzyme, indicate that the quenching of protein fluorescence of liver alcohol dehydrogenase by either ternary complex formation or alkaline pH is due to resonance energy transfer to tyrosinate. Likely candidates as energy acceptors are the Tyr-286 residues are within transfer distance for each Trp-314 residue, as well as being at the surface of the enzyme and 30 A from the active center zinc atom. Alkaline pH directly ionizes this tyrosine residue, while ternary complex formation causes a conformational change resulting in its ionization.

Spectral evidence for tyrosine ionization linked to a conformational change in liver alcohol dehydrogenase ternary complexes.

Resonance energy transfer from Trp-314 to ionized Tyr-286 was proposed (Laws, W. R., and Shore, J. D. (1978) J. Biol. Chem. 253, 8593-8597) as the mechanism for the observed decrease in protein fluorescence of liver alcohol dehydrogenase seen with alkaline pH, or with the formation of a ternary complex with NAD+ and trifluoroethanol. In the present study, ultraviolet difference spectra confirm the presence of ionized tyrosine not only in these two cases but also in the ternary complex with NADH and isobutyramide. Our results indicate that ternary complex formation, with either oxidized or reduced coenzyme, causes a conformational change leading to partial ionization of tyrosine residues in regions of the enzyme far from the active site.

Neurophysin-neurohypophyseal hormone interactions: studies using a dansylated vasotocin analogue.

We have synthesized a neurohypophyseal hormone analogue containing an extrinsic fluorescence probe by linking a dansyl (DNS) group to the epsilon-amino group of the lysine at residue 8 of vasotocin. The fluorescence properties of this analogue have been characterized by steady-state and time-resolved spectroscopic methods and compared with those of epsilon-DNS-lysine and the dansylated carboxyl terminal tripeptide Pro-Lys(DNS)-GlyNH2. The binding of this hormone analogue to purified isoforms of bovine neurophysins, the natural carrier proteins of the neurohypophyseal hormones, results in changes in several fluorescence parameters of the dansyl probe. These changes include an increase in intensity and average lifetime, a shift of the emission band to higher energies, and an increase in the emission anisotropy. Anisotropy changes have been used to determine dissociation constants for binding to these neurophysin isoforms. Based on the changes in the fluorescence properties of the dansyl probe, the dansyl group itself interacts with the protein. The degree of the dansyl-neurophysin interaction, however, appears to be different for the full sequence isoform of neurophysin I and the Val89 isoform of neurophysin II.

Fluorescence properties of a new guanosine analog incorporated into small oligonucleotides.

The fluorescence properties of 3-methyl-isoxanthopterin (3-MI) incorporated into different oligonucleotides have been determined. This highly fluorescent guanosine analog has its absorption and fluorescence spectra well resolved from those of the normal nucleotides and the aromatic amino acids. The small shifts observed in absorption and fluorescence emission spectra upon incorporation of 3-MI into these oligonucleotides are consistent with a general solvent effect and do not suggest any contribution from the position of the probe from the 5' end, the sequence of nucleotides immediately 5' or 3' to the probe, or the single- or double-stranded nature of the oligomer. However, steady-state and time-resolved fluorescence studies indicate that the presence of a purine immediately 5' or 3' to the probe results in some dynamic but mostly static quenching in the single-stranded oligomer. Furthermore, a 3' purine is more effective than a 5' purine, and an adenine appears to be more effective than a guanine for these static quenching interactions. Formation of the double-stranded oligomer leads to an additional loss of quantum yield, which can also be ascribed primarily to static quenching. These results show that this new class of spectrally enhanced fluorescent purine analogs will be able to provide useful information concerning the perturbation of nucleic acid structures.

Dynamics of biomolecules: assignment of local motions by fluorescence anisotropy decay.

Many biological systems have multiple fluorophores that experience multiple depolarizing motions, requiring multiple lifetimes and correlation times to define the fluorescence intensity and anisotropy decays, respectively. To simplify analyses, an assumption often made is that all fluorophores experience all depolarizing motions. However, this assumption usually is invalid, because each lifetime is not necessarily associated with each correlation time. To help establish the correct associations and recover accurate kinetic parameters, a general kinetic scheme that can examine all possible associations is presented. Using synthetic data sets, the ability of the scheme to discriminate among all nine association models possible for two lifetimes and two correlation times has been evaluated. Correct determination of the association model, and accurate recovery of the decay parameters, required the global analysis of related data sets. This general kinetic scheme was then used for global analyses of liver alcohol dehydrogenase anisotropy data sets. The results indicate that only one of the two tryptophan residues in each subunit is depolarized by process(es) independent of the enzyme's rotations. By applying the proper kinetic scheme and appropriate analysis procedures to time-resolved fluorescence anisotropy data, it is therefore possible to examine the dynamics of specific portions of a macromolecule in solution.

Human factor VIIa and its complex with soluble tissue factor: evaluation of asymmetry and conformational dynamics by ultracentrifugation and fluorescence anisotropy decay methods.

Ultracentrifugation and fluorescence anisotropy decay measurements were used to evaluate the asymmetry and conformational dynamics of human blood clotting enzyme VIIa (VIIa) and the complex it forms with a soluble truncation mutant of human tissue factor (sTF) which acts as an essential cofactor for VIIa. Sedimentation velocity experiments showed that both VIIa and the sTF.VIIa complex are highly asymmetric. In each case, the friction ratio f/fsphere, is consistent with a family of general elliposids ranging from prolate to oblate. Fluorescence anisotropy decay experiments were used to limit the family of elliposids which can describe the hydrodynamic behavior of VIIa and sTF.VIIa. For both VIIa and the sTF.VIIa complex, the oblate ellipsoid of revolution was eliminated. In addition, the fluorescence anisotropy decay data clearly show that upon binding sTF.VIIa loses a segmental motion involving a domain containing the active site of the enzyme. This suggests that sTF causes a stabilization of a limited range of VIIa conformations. This stabilization may be important for proper recognition of the TF.VIIa substrate, factor X.

Time-resolved fluorescence and 1H NMR studies of tyrosyl residues in oxytocin and small peptides: correlation of NMR-determined conformations of tyrosyl residues and fluorescence decay kinetics.

Steady-state and time-resolved fluorescence properties of the single tyrosyl residue in oxytocin and two oxytocin derivatives at pH 3 are presented. The decay kinetics of the tyrosyl residue are complex for each compound. By use of a linked-function analysis, the fluorescence kinetics can be explained by a ground-state rotamer model. The linked function assumes that the preexponential weighting factors (amplitudes) of the fluorescence decay constants have the same relative relationship as the 1H NMR determined phenol side-chain rotamer populations. According to this model, the static quenching of the oxytocin fluorescence can be attributed to an interaction between one specific rotamer population of the tyrosine ring and the internal disulfide bridge.

Correlation of tryptophan fluorescence intensity decay parameters with 1H NMR-determined rotamer conformations: [tryptophan2]oxytocin.

While the fluorescence decay kinetics of tyrosine model compounds [Laws, W. R., Ross, J. B. A., Wyssbrod, H. R., Beechem, J. M., Brand, L., & Sutherland, J. C. (1986) Biochemistry 25, 599-607] and the tyrosine residue in oxytocin [Ross, J. B. A., Laws, W. R., Buku, A., Sutherland, J. C., & Wyssbrod, H. R. (1986) Biochemistry 25, 607-612] can be explained in terms of heterogeneity derived from the three ground-state chi 1 rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from chi 2 rotations. In this paper, the time-resolved and steady-state fluorescence properties of [tryptophan2]oxytocin at pH 3 are presented and compared with 1H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength and a global analysis of these decay curves for common emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the 1H NMR-determined chi 1 rotamer populations of the indole side chain. 15N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the chi 1 rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two chi 2 populations. Depending upon the degree of correlation between chi 2 and chi 1, there may be from three to six side-chain conformations for the tryptophan residue. The combined fluorescence and NMR results are consistent with a rotamer model in which either (i) the chi 2 rotations are fast compared to the fluorescence intensity decay of the tryptophan residue, (ii) environmental factors affecting fluorescence intensity decay properties are dominated by chi 1 interactions, or (iii) the chi 2 and chi 1 rotations are highly correlated.

Time-resolved fluorescence and 1H NMR studies of tyrosine and tyrosine analogues: correlation of NMR-determined rotamer populations and fluorescence kinetics.

The time-resolved fluorescence properties of phenol and straight-chained phenol derivatives and tyrosine and simple tyrosine derivatives are reported for the pH range below neutrality. Phenol and straight-chained phenol derivatives exhibit single exponential fluorescence decay kinetics in this pH range unless they have a titratable carboxyl group. If a carboxyl group is present, the data follow a two-state, ground-state, Henderson-Hasselbalch relationship. Tyrosine and its derivatives with a free carboxyl group display complex fluorescence decay behavior as a function of pH. The complex kinetics cannot be fully explained by titration of a carboxyl group; other ground-state processes are evident, especially since tyrosine analogues with a blocked carboxyl group are also multiexponential. The fluorescence kinetics can be explained by a ground-state rotamer model. Comparison of the preexponential weighting factors (amplitudes) of the fluorescence decay constants with the 1H NMR determined phenol side-chain rotamer populations shows that tyrosine derivatives with a blocked or protonated carboxyl group have at least one rotamer exchanging more slowly than the radiative and nonradiative rates, and the fluorescence data are consistent with a slow-exchange model for all three rotamers, the shortest fluorescence decay constant is associated with a rotamer where the carbonyl group can contact the phenol ring, and in the tyrosine zwitterion, either rotamer interconversion is fast and an average lifetime is seen or rotamer interconversion is slow and the individual fluorescence decay constants are similar.

Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.

We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.

5-Hydroxytryptophan: an absorption and fluorescence probe which is a conservative replacement for [A14 tyrosine] in insulin.

Use of insulin's intrinsic tyrosine absorption and fluorescence to monitor its interaction with the insulin receptor is limited because the spectral properties of the receptor tryptophan residues mask the spectral properties of the hormone tyrosine residues. We describe the synthesis of an insulin analog where A14 tyrosine is replaced by a tryptophan analog, 5-hydroxytryptophan. This insulin is spectrally enhanced since 5-hydroxytryptophan has an absorption band above 300 nm which is at lower energies than the absorption of tryptophan. Steady-state and time-resolved fluorescence parameters indicate that 5-hydroxytryptophan reports the same information about the environment of the A14 side chain as does the corresponding tryptophan-containing insulin. The synthetic hormone is a full agonist compared to porcine insulin, but has slightly reduced specific activity. Consequently, this spectrally enhanced insulin analog will be useful for hormone-receptor interaction studies since it can be observed by both absorption and fluorescence even in the presence of the tryptophan-containing receptor.

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen.

Animals vaccinated with heat shock protein (HSP)--peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP--peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96--peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide--gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide--gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.