Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (beta 108Asn --> Lys).

HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive charge (per alpha beta dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the beta beta cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the beta-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2, 3-diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pathway linked to the His residues of the beta beta cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the beta beta cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.

Rapid loop dynamics of Yersinia protein tyrosine phosphatases.

The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand-free, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with chi2,1 dihedral angles of -4 degrees and -90 degrees. The two rotamers have been attributed to the presence of both "closed" and "open" WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate-ligated C403S PTPase contain a single W3 band which is correlated to the -4 degrees rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of approximately 2.6 x 10(8) s(-1). We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.

Folding of cytochrome c initiated by submillisecond mixing.

Cytochrome c folding was initiated using a new solution mixer that provides a time window which covers over 90% of the burst phase unresolved by conventional stop-flow measurements. Folding was followed by resonance Raman scattering. Kinetic analysis of the high frequency Raman data indicates that a nascent phase occurs within the mixing dead time of 100 microseconds. A significant fraction of the protein was found to be trapped in a misfolded bis-histidine form during the nascent phase at pH 4.5, thereby preventing the protein from folding rapidly and homogeneously. The nascent phase was followed by a haem-ligand exchange phase that populates the native histidine-methionine coordinated form through a thermodynamically controlled equilibrium.

Probing the hemoglobin central cavity by direct quantification of effector binding using fluorescence lifetime methods.

Time-resolved fluorescence methods have been used to show that 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), a fluorescent analog of 2,3-diphosphoglycerate, binds to the central cavity of carboxyhemoglobin A (HbACO) at pH 6.35. A direct quantitative approach, based on the distinctive free and bound HPT fluorescent lifetimes of 5.6 ns and approximately 27 ps, respectively, was developed to measure the binding affinity of this probe. HPT binds to a single site and is displaced by inositol hexaphosphate at a 1:1 mol ratio, indicating that binding occurs at the 2,3-diphosphoglycerate site in the central cavity. Furthermore, the results imply that low pH HbACO exists as an altered R state and not an equilibrium mixture of R and T states. The probe was also used to monitor competitive effector binding and to compare the affinity of the binding site in several cross-bridged HbA derivatives.

Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor.

To further our understanding of the protein folding process, it is desirable to examine the structural intermediates (equilibrium and kinetic) that are populated between the statistical coil state and the folded molecule. X-ray crystallography and NMR structural studies are unable to determine long-range distances in proteins under denaturing solution conditions. Nonradiative (Förster) energy transfer, however, has been shown to be a spectroscopic ruler for the measurement of distance distributions and diffusion between selected sites in proteins under a range of different solution conditions. The distributions of distances between a donor probe at the N-terminal residue and an acceptor attached to one of the four lysine residues (15, 26, 41, 46) of reduced and unfolded (in 6 M guanidine hydrochloride and 20 mM dithiothreitol) bovine pancreatic trypsin inhibitor (BPTI) were measured as a function of temperature. Even in strong denaturant and reducing agent, BPTI does not exist as a statistical coil polypeptide. It appears that nonlocal (long-range) interactions are already beginning to "fold" the protein toward a more compact, native conformation. As the temperature is increased under these conditions, hydrophobic interactions lead to an even more compact structure consistent with the predictions of phase diagrams for globular proteins.

Stark effect spectroscopy of carotenoids in photosynthetic antenna and reaction center complexes.

The effects of electric fields on the absorption spectra of the carotenoids spheroidene and spheroidenone in photosynthetic antenna and reaction center complexes (wild-type and several mutants) from purple non-sulfur bacteria are compared with those for the isolated pigments in organic glasses. In general, the field effects are substantially larger for the carotenoid in the protein complexes than for the extracted pigments and larger for spheroidenone than spheroidene. Furthermore, the electrochromic effects for carotenoids in all complexes are much larger than those for the Qx transitions of the bacteriochlorophyll and bacteriopheophytin pigments which absorb in the 450-700 nm spectral region. The underlying mechanism responsible for the Stark effect spectra in the complexes is found to be dominated by a change in permanent dipole moment of the carotenoid upon excitation. The magnitude of this dipole moment change is found to be considerably larger in the B800-850 complex compared to the reaction center for spheroidene; it is approximately equivalent in the two complexes for spheroidenone. These results are discussed in terms of the effects of differences in the carotenoid functional groups, isomers and perturbations on the electronic structure from interactions with the organized environment in the proteins. these data provide a quantitative basis for the analysis of carotenoid bandshifts which are used to measure transmembrane potential, and they highlight some of the pitfalls in making such measurements on complex membranes containing multiple populations of carotenoids. The results for spheroidenone should be useful for studies of mutant proteins, since mutant strains are often grown semi-aerobically to minimize reversion.

Large protein-induced dipoles for a symmetric carotenoid in a photosynthetic antenna complex.

Unusually large electric field effects have been measured for the absorption spectra of carotenoids (spheroidene) in the B800-850 light-harvesting complex from the photosynthetic bacterium Rhodobacter sphaeroides. Quantitative analysis shows that the difference in the permanent dipole moment between the ground state and excited states in this protein complex is substantially larger than for pure spheroidene extracted from the protein. The results demonstrate the presence of a large perturbation on the electronic structure of this nearly symmetric carotenoid due to the organized environment in the protein. This work also provides an explanation for the seemingly anomalous dependence of carotenoid band shifts on transmembrane potential and a generally useful approach for calibrating electric field-sensitive dyes that are widely used to probe potentials in biological systems.