Biomedical subjects

R Leenders

Publications and source records attributed to R Leenders.

3 recordsLinked to original sources

PubMed1994

Molecular dynamics simulations of oxidized and reduced Clostridium beijerinckii flavodoxin.

Molecular dynamics simulations of oxidized and reduced Clostridium beijerinckii flavodoxin in water have been performed in a sphere of 1.4-nm radius surrounded by a restrained shell of 0.8 nm. The flavin binding site, comprising the active site of the flavodoxin, was in the center of the sphere. No explicit information about protein-bound water molecules was included. An analysis is made of the motional characteristics of residues located in the active site. Positional fluctuations, hydrogen bonding patterns, dihedral angle transitions, solvent behavior, and time-dependent correlations are examined. The 375-ps trajectories show that both oxidized and reduced protein-bound flavins are immobilized within the protein matrix, in agreement with earlier obtained time-resolved fluorescence anisotropy data. The calculated time-correlated behavior of the tryptophan residues reveals significant picosecond mobility of the tryptophan side chain located close to the reduced isoalloxazine part of the flavin.

Binding Sites↗

PubMed1993

Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time-resolved polarized fluorescence.

The time-resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii have been examined. The fluorescence intensity decays were analyzed using the maximum-entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein-bound FMN. Three fluorescence lifetime components are found in oxidized flavodoxin, two of which are not present in the fluorescence-intensity decay of free FMN. The main component is distributed at 30 ps, with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluorescence decay of free FMN. The results allowed us to determine the dissociation constant, Kd = 2.61 x 10(-10) M (at 20 degrees C). Collisional fluorescence-quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, indicating that it possibly originates from an active-site conformation in which the flavin is more or less buried in the protein and not accessible to iodide. The fluorescence anisotropy behaviour of free and protein-bound FMN was examined and analyzed with the maximum-entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein-bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution. Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about 1 ns. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry 11, 4746-4754] in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN. The two shortest fluorescence lifetimes, which do not carry information on the long-term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to overall protein tumbling. These lifetime components probably originate from a complex in which the flavin-ring system is more or less immobilized within the protein matrix.

Clostridium↗

PubMed1993

Flavin dynamics in reduced flavodoxins. A time-resolved polarized fluorescence study.

The time-resolved fluorescence and fluorescence anisotropy characteristics of reduced flavin mononucleotide in solution as well as bound in flavodoxins isolated from the bacteria Desulfovibrio gigas, Desulfovibrio vulgaris, Clostridium beijerinckii MP and Megasphaera elsdenii have been examined. All fluorescence and fluorescence anisotropy decays were analyzed by two different methods: (a) least-squares fitting with a sum of exponentials and (b) the maximum entropy method to yield distributed lifetimes and correlation times. The results of both approaches are in excellent agreement. The fluorescence decay of the free as well as protein-bound reduced flavin chromophore is made up of three components. The shortest component proves to be relatively sensitive to the environment and can therefore be used as a diagnostic tool to probe the microenvironment of the reduced isoalloxazine ring system. The other two longer fluorescence lifetime components are insensitive to the chromophore environment and seem therefore to be related to intrinsic, photophysical properties of the reduced chromophore. Fluorescence anisotropy decays show that the flavin mononucleotide in all four reduced flavodoxins is immobilized within the protein matrix, as indicated by the recovery of a single rotational correlation time, reflecting the rotation of the whole protein. No indications are found that rapid structural fluctuations occur in reduced flavodoxins, and the mechanism of electron transfer from flavodoxin to other redox proteins seems to involve immobilized reduced flavin.

Clostridium↗