'In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the ... More
Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies.
AuthorsZhang Z, Spiering MM, Trakselis MA, Ishmael FT, Xi J, Benkovic SJ, Hammes GG
JournalProc Natl Acad Sci U S A
PubMed ID15728347
'Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with ... More
Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase.
'Ensemble kinetics and single-molecule fluorescence microscopy were used to study conformational transitions associated with enzyme catalysis by dihydrofolate reductase (DHFR). The active site loop of DHFR was labeled with a fluorescence quencher, QSY35, at amino acid position 17, and the fluorescent probe, Alexa555, at amino acid 37, by introducing cysteines ... More
Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.
AuthorsDeRocco V, Anderson T, Piehler J, Erie DA, Weninger K,
JournalBiotechniques
PubMed ID21091445
To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to ... More
Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism.
AuthorsMcGeoch AT, Trakselis MA, Laskey RA, Bell SD
JournalNat Struct Mol Biol
PubMed ID16116441
The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the ... More
Optimizing methods to recover absolute FRET efficiency from immobilized single molecules.
Microscopy-based fluorescence resonance energy transfer (FRET) experiments measure donor and acceptor intensities by isolating these signals with a series of optical elements. Because this filtering discards portions of the spectrum, the observed FRET efficiency is dependent on the set of filters in use. Similarly, observed FRET efficiency is also affected ... More
Single-molecule fluorescence studies of a PH domain: new insights into the membrane docking reaction.
AuthorsKnight JD, Falke JJ,
JournalBiophys J
PubMed ID19167305
Proteins containing membrane targeting domains play essential roles in many cellular signaling pathways. However, important features of the membrane-bound state are invisible to bulk methods, thereby hindering mechanistic analysis of membrane targeting reactions. Here we use total internal reflection fluorescence microscopy (TIRFM), combined with single particle tracking, to probe the ... More
Nuclear import time and transport efficiency depend on importin beta concentration.
AuthorsYang W, Musser SM
JournalJ Cell Biol
PubMed ID16982803
Although many components and reaction steps necessary for bidirectional transport across the nuclear envelope (NE) have been characterized, the mechanism and control of cargo migration through nuclear pore complexes (NPCs) remain poorly understood. Single-molecule fluorescence microscopy was used to track the movement of cargos before, during, and after their interactions ... More
Prion recognition elements govern nucleation, strain specificity and species barriers.
AuthorsTessier PM, Lindquist S
JournalNature
PubMed ID17495929
Prions are proteins that can switch to self-perpetuating, infectious conformations. The abilities of prions to replicate, form structurally distinct strains, and establish and overcome transmission barriers between species are poorly understood. We exploit surface-bound peptides to overcome complexities of investigating such problems in solution. For the yeast prion Sup35, we ... More
Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations.
AuthorsWeninger K, Bowen ME, Chu S, Brunger AT
JournalProc Natl Acad Sci U S A
PubMed ID14657376
Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, ... More