Michael K. Helms

Time-resolved fluorescence studies on site-directed mutants of human serum albumin.

Human serum albumin (HSA) contains a single tryptophan residue at position 214. The emission properties of tryptophan 214 from recombinant albumins, namely, normal HSA, FDH‐HSA and a methionine 218 HSA were examined. In all cases, the excited state lifetimes were best described by a two component model consisting mainly of a Lorentzian distribution. The centers of these distributions were 5.60 ns for HSA, 4.23 ns for FDH‐HSA, and 6.08 ns for Met‐218 HSA. The global rotational correlation times of the three HSAs were near 41 ns while the amplitude and rate of the local motion varied. These changes in the lifetimes and mobilities suggest perturbation in the local protein environment near tryptophan 214 as a consequence of the amino acid substitutions.

Correlation Between Self-Association Modes and GTPase Activation of Dynamin

The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 × 1017 M−3. Stopped-flow fluorescence experiments show that the association rate constant for 2′(3′)-O-N-methylanthraniloyl (mant)GTP is 7.0 × 10−5 M−1 s−1 and the dissociation rate constant is 2.1 s−1, whereas the dissociation rate constant for mantdeoxyGDP is 93 s−1. We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.

Ground-and Excited-State Characterization of an Electrostatic Complex between Tetrakis-(4-Sulfonatophenyl)porphyrin and 16-Pyrimidinium Crown-4

Abstract— We report the formation of an electrostatic complex between (16‐pyrimidinium crown‐4)tetranitrate (16PC4) and tetrakis‐(4‐sulfonatophenyl)porphyrin (4SP) in aqueous solution. Ground‐state complex formation results in a red shift of the 4SP visible absorption bands and a decrease in absorbance of the Soret band. The equilibrium constant for complex formation (determined from optical titrations) is found to be (2.0 ± 0.2) × 105M−1. In addition, the data fit to an expression describing a 1:1 stoichiometry. Excitation of the complex results in quenching of both the excited singlet and triplet states of the associated porphyrin. The singlet‐state lifetime decreases from 10 ns for the free porphyrin to 1.5 ns in the presence of 16PC4 at low solution ionic strengths. In addition, evidence is presented for triplet‐state quenching within the complex with kq= (1.1 ± 0.1) × 104 s−1. The mechanism of quenching is tentatively assigned to electron transfer from either the excited singlet or excited triplet state of the porphyrin to the ground state of the 16PC4.

Conformational dynamics and temperature dependence of photoinduced electron transfer within self-assembled coproporphyrin:cytochrome c complexes.

The focus of the present study is to better understand the complex factors influencing intermolecular electron transfer (ET) in biological molecules using a model system involving free-base coproporphyrin (COP) complexed with horse heart cytochrome c (Cc). Coproporphyrin exhibits bathochromic shifts in both the Soret and visible absorption bands in the presence of Cc and an absorption difference titration reveals a 1:1 complex with an association constant of 2.63 +/- 0.05 x 10(5) M(-1). At 20 degrees C, analysis of time-resolved fluorescence data reveals two lifetime components consisting of a discrete lifetime at 15.0 ns (free COP) and a Gaussian distribution of lifetimes centered at 2.8 ns (representing (1)COP --> Cc ET). Temperature-dependent, time-resolved fluorescence data demonstrate a shift in singlet lifetime as well as changes in the distribution width (associated with the complex). By fitting these data to semiclassical Marcus theory, the reorganizational energy (lambda) of the singlet state electron transfer was calculated to be 0.89 eV, consistent with values for other porphyrin/Cc intermolecular ET reactions. Using nanosecond transient absorption spectroscopy the temperature dependences of the forward and thermal back ET originating from triplet state were examined ((3)COP --> Cc ET). Fits of the temperature dependence of the rate constants to semiclassical Marcus theory gave lambda of 0.39 eV and 0.11 eV for the forward and back triplet ET, respectively (k(f) = (7.6 +/- 0.3) x 10(6) s(-1), k(b) = (2.4 +/- 0.3) x 10(5) s(-1)). The differing values of lambda for the forward and back triplet ET demonstrate that these ET reactions do not occur within a static complex. Comparing these results with previous studies of the uroporphyrin:Cc and tetrakis (4-carboxyphenyl)porphyrin:Cc complexes suggests that side-chain flexibility gives rise to the conformational distributions in the (1)COP --> Cc ET whereas differences in overall porphyrin charge regulates gating of the back ET reaction (reduced Cc --> COP(+)).

Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli

Elongation factor Tu from Escherichia coli is known to polymerize at slightly acidic pH and low ionic strength. The structure and dynamics of these aggregates have been examined using imaging and spectroscopic methodologies. Electron microscopy provides evidence for two-dimensional sheets and bundled filaments of EF-Tu, whereas fluorescence microscopy of EF-Tu covalently labeled with tetramethylrhodamine isothiocyanate showed highly branched polymers of EF-Tu several microns in diameter. These polymers were studied using quasi-elastic light scattering to determine the evolution of the translational diffusion coefficient during the polymerization process. The rotational dynamics of the aggregate were investigated using phosphorescence anisotropy of EF-Tu covalently labeled with erythrosin isothiocyanate. A high infinite-time anisotropy was observed, suggesting a lack of motion or entanglement of EF-Tu polymers. A sub-microsecond motion which was slowed in the presence of glycerol may be due to local flexibility of the polymers. The possible relevance of polymeric EF-Tu to its function in vivo is discussed.

Spectroscopic Characterization of Two Soluble Transducers from the Archaeon Halobacterium salinarum

In the present study, structural aspects of the two soluble transducers, HtrX and HtrXI, from the archaeon H. salinarum have been examined using UV circular dichroism and steady-state fluorescence spectroscopies. Circular dichroism (CD) data indicate that both HtrX and HtrXI exhibit salt-dependent protein folding. Under low-ionic-strength conditions (0.2 M NaCl or KCl) the CD spectra of HtrXI is similar to that of the Gdn-HCl- or urea-denatured forms and is indicative of random coil structure. In contrast, the CD spectrum of HtrX under low-ionic-strength conditions contains roughly 85% α-helical character, indicating a significant degree of folding. Addition of NaCl or KCl to solutions of HtrX or HtrXI results in CD features consistent with predominately α-helical character (>95%) for both proteins. In addition, the transition points (i.e., ionic strengths at which the protein converts from random coil to α-helical character) are quite distinct and dependent upon the type of salt present (i.e., either NaCl or KCl). Accessibility of tryptophan residues to the solvent was also examined for both HtrX and HtrXI in both folded and unfolded states using Kl quenching. The Stern–Volmer constants obtained suggest that the tryptophans (Trp35 in HtrX and both Trp47 and Trp74 in HtrXI) are partially exposed to the solvent, indicating that they are located near the surface of the protein in all three cases. Furthermore, fluorescence quenching with the single Trp mutants Trp74AIa and Trp47AIa of HtrXI indicates different environments for these two residues.

Temperature dependence of photoinduced electron transfer within self-associated porphyrin: guanine monophosphate complexes

Abstract In the current report, the temperature dependence of photoinduced electron transfer between tetrakis-(4-tetramethylpyridyl)porphine (T4MPyP) and guanine monophosphate (GMP) has been examined. In the presence of GMP the fluorescence lifetime analysis reveals a Lorentzian distribution of lifetimes centered at 0.7 ns with a width of 0.9 ns displaying significant temperature dependence. Fitting temperature dependent data to the Marcus equation gives a reorganizational energy ( λ ) for the electron transfer reaction of 0.6 eV and an electronic coupling factor ( H AB ) of 3×10 −3 eV . These results suggest conformational regulation of electron transfer within the non-covalent porphyrin:nucleotide complex.

FLEXIBILITY INVOLVING THE INTERMOLECULAR DITYROSYL CROSS-LINKS OF ENZYMATICALLY POLYMERIZED CALMODULIN

The role of dityrosine as a fluorescent crossbridge between adjacent calmodulin molecules within the high molecular mass polymers that are generated by Arthromyces peroxidase-catalyzed cross-linking [Malencik, D. A., and Anderson, S. R. (1996) Biochemistry 35, 4375] has been examined in frequency domain fluorescence anisotropy studies. Measurements on a polymer fraction possessing a range of molecular masses > 96 000 in NaDodSO4 polyacrylamide gel electrophoresis demonstrate predominating fast local rotations involving the dityrosyl moieties. Normal distribution analyses of the results show peak rotational correlation times of 0.6 ns (zero Ca2+) and 1.2 ns (+Ca2+), values that are smaller than the principal correlation times determined for the global rotation of the free calmodulin monomer in either the presence or absence of Ca2+. The intermolecularly cross-linked segments of the polymers retain a degree of the mobility that is characteristic of the tyrosine-containing sequences of native calmodulin. The half-widths of the normal distribution curves range from 13 ns (zero Ca2+) to approximately 90 ns (5 mM Ca2+), thus encompassing varying rates of segmental motion within the polymers. When Ca2+ is present, possible contributions from the global rotations of polymer molecules are detected near the operating limits of the method. Experiments with the intramolecularly cross-linked calmodulin monomer give global rotational correlation times of 7.9 ns (zero Ca2+) and 11.4 ns (+Ca2+), which compare to values of 7.2 ns and 9.9 ns found previously in time domain measurements [Small, E. W., and Anderson, S. R. (1988) Biochemistry 27, 419]. Rotations of apparent phi2 = 0.2 to 0.3 ns also are detected, accounting for 31% (-Ca2+) to 23% (+Ca2+) of the anisotropy.