Lars Edman

The fluctuating enzyme: a single molecule approach

Abstract The excess of structural degrees of freedom in a protein enzyme opens questions about the conformational homogeneity. We studied single horseradish peroxidase enzyme turnovers by fluorescence spectroscopy. Application of a two-state dynamic model to the measured data shows exponential product dissociation kinetics, but a large distribution of rates for the enzyme to form the enzyme-product complex. The experiments show that in addition to the peroxidative cycle thermodynamic fluctuation phenomena on a wide range of time scales affect enzyme activity.

NON-ERGODIC BEHAVIOUR IN CONFORMATIONAL TRANSITIONS OF SINGLE DNA MOLECULES

Abstract Conformational fluctuations in single nucleic acid molecules have recently been observed through excited state lifetime measurements. Immobilisation of the sample molecule has also enabled direct observation of the fluorescence intensity fluctuations generated as the molecule switches between two conformations. As a probe for conformational fluctuations we use tetramethylrhodamine linked to a 217-bp DNA oligonucleotide. The measurements on this and similar systems reveal the existence of a distribution of reaction rates between the conformations. Here we report 37 detected single-molecule conformational fluctuations collected with the same immobilisation method as described earlier. Within the time window of observation the reaction rates differ between the molecules, but stay constant within a single molecule. The distribution of the relaxation rates between the molecules correspond to the distribution seen in a bulk measurement on a similar system. We therefore conclude that within the observation time window the single DNA molecules behave in a non-ergodic way.

Heterogeneity in single DNA conformational fluctuations

Abstract Non-exponential behaviour in bulk measurements of two-state molecular reactions can have different origins. The probability density for the time which one of two states is occupied during different observation interval times can be used to reveal system characteristics if single molecules are studied. An evaluation of new experimental data from single DNA molecules labeled with a fluorescent conformation probe proves the heterogeneity of the ensemble system. Also, the anomalicity is concluded to be low for single events. The evaluation method can be applied in other experiments where two-state reversible processes are studied for isolated systems.