Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about conformational changes in biological macromolecules, including the reaction pathways and rearrangements involved in sequence-specific DNA ‘breathing’ and the assembly of protein-nucleic acid complexes. Because microsecond single-molecule trajectories often involve ‘sparse’ data – i.e., they contain relatively few data points per unit time – they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high ‘data density.’ We here describe a generalized approach, based on time correlation functions (TCFs), to obtain kinetic information from microsecond single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on the reaction pathways connecting relatively long-lived reactant and product states. We have applied the generalized TCF approach to a specific, biologically relevant example drawn from studies of the assembly mechanisms of the single-stranded (ss) DNA binding protein (gp32) of the T4 bacteriophage replication complex onto a model DNA replication fork. We performed microsecond single-molecule Förster resonance energy transfer (smFRET) measurements of Cy3/Cy5 labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 chromophores, which label the ends of a short (15 nucleotide) segment of ssDNA, and permit us to track the stochastic inter-conversions between various protein-bound and unbound configurations of the ssDNA. The results of our analysis reveal the presence of short-lived intermediates, which play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks.

Using multi-order Time Correlation Functions (TCFs) to elucidate biomolecular reaction pathways from microsecond single-molecule fluorescence experiments

253rd National Meeting (2017)

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