USE OF A PULSED LASER DIODE TO MEASURE PICOSECOND FLUORESCENCE LIFETIMES

Abstract— The use of an inexpensive pulsed laser diode (Hamamatsu picosecond light pulser PLP-01) as the excitation source for a single photon timing spectrolluorimeter with microchannel plate photomultiplier detection was dem-onstrated. The performance of the instrument was tested with two very short-lived fluorescent dyes and two pho-tosynthetic systems with wcll-defined decay characteristics. Individual fluorescence decays were analyzed by modeling with a convolution of the instrument response function to a sum of exponential decay components. Accurate fluorcscence lifetimcs of the dyes cryptocyanine (55 ps in acetone and 83 ps in ethanol) and 1,1'-diethyl-2,2'-dicarbocyanine iodide (13 ps in acetone and 26 ps in ethanol) were obtained by analysis of the decay kinetics with a single exponential component. Fits to the fluorescence decay kinetics of isolated photosystem I particles and intact cyanobacterial cells required three and four decay components. respectively. The decay kinetics of the isolated photosystem I preparation were dominated (99%) by a very fast 9 ps lifetime, reflecting the preparation's small antenna size of approximately 30 chlorophyll a . The cyanobackria showed decay components of 35 ps, 160 ps, 400 ps and 1.95 ns similar to those described previously by Mullincaux and Holzwarth ( Rinchim. Biophys. Acfa 1098 , 68–78, 1991). The performance of the pulsed laser diode as an excitation source for single photon timing is discussed in comparison with conventional sources of picosecond light pulses.     

Enzymes do what is expected (chalcone isomerase versus chorismate mutase)

Madicago sativa chalcone isomerase (CI) catalyzes the isomerization of chalcone to flavanone, whereas E. coli chorismate mutase (CM) catalyzes the pericyclic rearrangement of chorismate to prephenate. Covalent intermediates are not formed in either of the enzyme-catalyzed reactions, K(M) and k(cat) are virtually the same for both enzymes, and the rate constants (k(o)) for the noncatalyzed reactions in water are also the same. This kinetic identity of both the enzymatic and the nonenzymatic reactions is not shared by a similarity in driving forces. The efficiency (DeltaG(o)() - DeltaG(cat)()) for the CI mechanism involves transition-state stabilization through general-acid catalysis and freeing of three water molecules trapped in the E.S species. The contribution to lowering DeltaG(cat)() by an increase in near attack conformer (NAC) formation in E.S as compared to S in water is not so important. In the CM reaction, the standard free energy for NAC formation in water is 8.4 kcal/mol as compared to 0.6 kcal/mol in E.S. Because the value of (DeltaG(o)() - DeltaG(cat)()) is 9 kcal/mol, the greater percentage of NACs accounts for approximately 90% of the kinetic advantage of the CM reaction. There is no discernible transition-state stabilization in the CM reaction. These results are discussed. In anthropomorphic terms, each enzyme has had to do what it must to have a biologically relevant rate of reaction.     

Just a near attack conformer for catalysis (chorismate to prephenate rearrangements in water,antibody, enzymes,and their mutants)

Standard free energies for formation of ground-state reactive conformers (DeltaGN degrees ) and transition states (DeltaG) in the conversion of chorismate to prephenate in water, B. subtilis mutase, E. coli mutase, and their mutants, as well as a catalytic antibody, are related by DeltaG = DeltaGN degrees + 16 kcal/mol. Thus, the differences in the rate constants for the water reaction and catalysts reactions reside in the mole fraction of substrate present as reactive conformers (NACs). These results, and knowledge of the importance of transition state stabilization in other cases, suggest a proposal that enzymes utilize both NAC and transition state stabilization in the mix required for the most efficient catalysis.     

A convenient synthesis of the cytidyl 3′-terminal monomer for solid-phase synthesis of RNG oligonucleotides

Replacing the phosphodiester backbone of RNA with positively charged guanidinium linkages has been shown to enable RNA oligomers to overcome electrostatic repulsion and bind double-stranded DNA in a triplex with high affinity. Ribonucleotide monomers with the ability to form guanidinium linkages have been synthesized for the generation of ribooligonucleotides with guanidinium linkages (RNGs) through solid-phase synthesis. We report herein an efficient method for the synthesis of N⁴-benzoyl-2′-O-(tert-butyldimethylsilyl)-5′-N-(4-monomethoxytritylamino)-3′-O-succinyl-5′-deoxycytidine, a new monomer required for the solid-phase synthesis of cytidyl RNG oligonucleotides.     

The mechanism of cis-trans isomerization of prolyl peptides by cyclophilin

The mechanism of cis-trans isomerization of prolyl peptides catalyzed by cyclophilin (CyP) was studied computationally via molecular dynamics (MD) simulations of the transition state (TS) and the cis and trans forms of the ground state (GS), when bound to CyP and when free in aqueous solution. The MD simulations include four enzyme-bound species of tetrapeptide (Suc-Ala-XC([double bond]O)-NPro-Phe-pNA; X = Gly, Trp, Ala, and Leu). In water, the prolyl amide bond is favorably planar with the presence of conformers exhibiting +/-20 degrees twist of the C-N dihedral. In the active site a hydrogen bond between the cis-prolyl amide carbonyl O and the backbone amide N-H of Asn102 retains the 20 degrees twist of the C-N dihedral. The TS structure is characterized by a 90 degrees twist of the amide C-N bond and a more favorable interaction with Asn102 due to the shorter distance between Asn102(HN) and the amide carbonyl O. The conformational change of cis --> TS also involves pyramidalization of the amide N, which results in the formation of a hydrogen bond between the amide N and the guanidino group of Arg55. Both Asn102 and Arg55 are held in the same position in CyP.cis-isomer as in CyP.TS. In the ligand-free CyP the Arg55 guanidino group is highly disorganized and Asn102 is displaced 1 A from the position in the ligand-bound CyP. Thus, the organization of Arg55 and Asn102 occurs upon substrate binding. The geometrical complimentarity of the organized enzyme structure to the TS structure is a result of preferential binding of the proline N and the amide carbonyl of the TS compared to that of GS. However, the N-terminal part (Suc-Ala) becomes repositioned in the TS such that two hydrogen bonds disappear, one hydrogen bond appears and two other hydrogen bonds becomes weaker on the conversion of CyP.cis to CyP.TS. During this conversion, total hydrophobic contact between enzyme and the peptide is preserved. Thus, the interaction energies of GS and TS with enzyme are, as a whole, much alike. This does not support the contention that TS is bound more tightly than GS by K(m)/K(TS) = 10(6) in the cis --> trans reaction. Repositioning of the N-terminal part of the peptide on CyP.TS formation becomes more pronounced when the substrate X residue is changed from Gly < Trp < Ala < Leu. We propose that the larger turning of the N-terminus is responsible for the larger value of the experimentally observed Delta S(++) and Delta H(++), which sum up to little change in Delta G(++). The positioning of the Arg55 and the degree of 20 degrees twist of the amide C-N bond are considered as criteria for Near Attack Conformers (NACs) in cis-trans isomerization. NACs account for approximately 30% of the total GS populations of the cis-isomer. Similar NAC populations were observed with four different substrates. This is consistent with the insensitivity of enzymatic activity to the nature of the X residue. Also, the NAC population in CyP.trans-AAPF was comparable to that in CyP.cis-AAPF, in accord with similar experimentally measured rates of the cis --> trans and trans --> cis reaction in CyP. These NACs, found in CyP.cis and CyP.trans, resemble only one of the four possible TS configurations in the water reaction. The identity of this TS structure (syn/exo) is in accord with experimentally determined KIE values in the enzymatic reaction. However, the geometry of the active site was also complementary to another TS structure (anti/exo) that was not detected in the active site by the same KIE measurements, implying that the geometrical fitness of the TS cannot be a single determining factor for enzymatic reactions.     

Dynamic structures of horse liver alcohol dehydrogenase (HLADH): results of molecular dynamics simulations of HLADH-NAD(+)-PhCH(2)OH, HLADH-NAD(+)-PhCH(2)O(-), and HLADH-NADH-PhCHO.

Molecular dynamics simulations of the oxidation of benzyl alcohol by horse liver alcohol dehydrogenase (HLADH) have been carried out. The following three states have been studied: HLADH.PhCH(2)OH.NAD(+) (MD1), HLADH.PhCH(2)O(-).NAD(+) (MD2), and HLADH.PhCHO.NADH (MD3). MD1, MD2, and MD3 simulations were carried out on one of the subunits of the dimeric enzyme covered in a 32-A-radius sphere of TIP3P water centered on the active site. The proton produced on ionization of the alcohol when HLADH.PhCH(2)OH.NAD(+) --> HLADH.PhCH(2)O(-).NAD(+) is transferred from the active site to solvent water via a hydrogen bonding network consisting of serine48 hydroxyl, ribose 2'- and 3'-hydroxyl groups, and Hist51. Hydrogen bonding of the 3'OH of ribose to Ile269 carbonyl maintains this proton in position to be transferred to water. Molecular dynamic simulations have been employed to track water1287 from the TIP3 water pool to the active site, thus exhibiting the mode of entrance of water to the active site. With time the water1287 accumulates in two different positions in order to accept the proton from the ribose 3'-OH and from His51. There can be identified two structural substates for proton passage. In the first substate the imidazole Ne2 of His51 is adjacent to the nicotinamide ribose C2'-OH and hydrogen bonding distances for proton transfer through the hydrogen bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...His51...OH(2) (path 1) average 2.0, 2.0, and 2.1 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A. The structure for path 1 is present 20% of the time span. And in the second substate, there are two possible proton passages: path 1 as before and path 2. Path 2 involves the hydrogen-bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...Ribose3'-OH...His51.OH(2) with the average bonding distances being 2.0, 2.0, 2.1, and 2.0 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A (20% probability of the time span), respectively. During the molecular dynamics simulation the NAD(+) ribose conformations have stabilized at the C2'-endo-C3'-exo or the C2'-endo conformations. With the C2'-endo conformation the first and second substates are able to persist for different time spans, while with the C2'-endo-C3'-exo conformation the only possible pathway involves the first substate. For both first and second substates the fluctuation of the distances between the ribose-OH protons and N epsilon 2 of His51 imidazole ring is partially contributed by the "windshield wiper" motion of the His51 imidazole ring. Since the imidazole of His-51 contributes only about 10-fold to activity, as estimated from the decrease in activity upon substitution with a Gln, there must be an alternate route for the proton to pass to solvent without going through this histidine. A third pathway involves ribose C3'-OH and Ile-269. In MD2, near attack conformers (NACs) for hydride transfer from PhCH(2)O(-) to NAD(+) represent approximately 60% of E.S conformers. The molecular dynamic study of MD3 at mildly basic pH reveals that reactive ground state conformers (NACs) for hydride transfer from NADH to PhCHO amount to 12 mol % of conformers. In MD3, anisotropic bending of the dihydronicotinamide ring of NADH (average value of alpha(c) = 4.0 degrees and alpha(n) = 0.5 degrees, respectively) is observed.     

Comparison of formation of reactive conformers (NACs) for the Claisen rearrangement of chorismate to prephenate in water and in the E. coli mutase: the efficiency of the enzyme catalysis

The Claisen rearrangements of chorismate (CHOR) in water and at the active site of E. coli chorismate mutase (EcCM) have been compared. From a total of 33 ns molecular dynamics simulation of chorismate in water solvent, seven diaxial conformers I-VII were identified. Most of the time (approximately 99%), the side chain carboxylate of the chorismate is positioned away from the ring due to the electrostatic repulsion from the carboxylate in the ring. Proximity of the two carboxylates, as seen in conformer I, is a requirement for the formation of a near attack conformer (NAC) that can proceed to the transition state (TS). In the EcCM.CHOR complex, the two carboxylates of CHOR are tightly held by Arg28 of one subunit and Arg11* of the other subunit, resulting in the side chain C16 being positioned adjacent to C5 with their motions restricted by van der Waals contacts with methyl groups of Val35 and Ile81. With the definition of NAC as the C5...C16 distance < or =3.7 A and the attack angle < or =30 degrees, it was estimated from our MD trajectories that the free energy of NAC formation is approximately 8.4 kcal/mol above the total ground state in water, whereas in the enzyme it is only 0.6 kcal/mol above the average of the Michaelis complex EcCM.CHOR. The experimentally measured difference in the activation free energies of the water and enzymatic reactions (Delta Delta G(++)) is 9 kcal/mol. It follows that the efficiency of formation of NAC (7.8 kcal/mol) at the active site provides approximately 90% of the kinetic advantage of the enzymatic reaction as compared to the water reaction. Comparison of the EcCM.TSA (transition state analogue) and EcCM.NAC simulations suggests that the experimentally measured 100 fold tighter binding of TSA compared to CHOR does not originate from the difference between NAC and the TS binding affinities, but might be due to the free energy cost to bring the two carboxylates of CHOR together to interact with Arg28 and Arg11* at the active site. The two carboxylates of TSA are fixed by a bicyclic structure. The remaining approximately 10% of Delta Delta G(++) may be attributed to a preferential interaction of Lys39-NH(3)(+) with O13 ether oxygen in the TS.