Biomedical subjects

James H Werner

Publications and source records attributed to James H Werner.

6 recordsLinked to original sources

PubMed2006

A two-dimensional view of the folding energy landscape of cytochrome c.

Time-correlated single photon counting (TCSPC) was combined with fluorescence correlation spectroscopy (FCS) to study the transition between acid-denatured states and the native structure of cytochrome c (Cyt c) from Saccharomyces cerevisiae. The use of these techniques in concert proved to be more powerful than either alone, yielding a two-dimensional picture of the folding energy landscape of Cyt c. TCSPC measured the distribution of distances between the heme of the protein and a covalently attached dye molecule at residue C102 (one folding reaction coordinate), whereas FCS measured the hydrodynamic radius (a second folding reaction coordinate) of the protein over a range of pH values. These two independent measurements provide complimentary information regarding protein conformation. We see evidence for a well defined folding intermediate in the acid renaturation folding pathway of this protein reflected in the distribution of lifetimes needed to fit the TCSPC data. Moreover, FCS studies revealed this intermediate state to be in dynamic equilibrium with unfolded structures, with conformational fluctuations into and out of this intermediate state occurring on an approximately 30-micros time scale.

Biophysics↗

PubMed2006

A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation.

We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one- (OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of micros and tens of micros due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of micros due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.

Biophysical Phenomena↗

PubMed2005

Site-specific dimensions across a highly denatured protein; a single molecule study.

Do highly denatured proteins adopt random coil configurations? Here, we address this question by measuring residue-to-residue separations across the denatured FynSH3 domain. Using single-molecule Forster resonance energy transfer techniques, we have collected transfer efficiency probability distributions for dye-labeled, denatured protein. Applying maximum likelihood analysis to the interpretation of these distributions, we have determined the through-space distance between five residue pairs in the protein's guanidine hydrochloride-unfolded and trifluoroethanol-unfolded states. We find that, while the dimensions of the guanidine hydrochloride -unfolded molecule generally coincide with the dimensions predicted for a random coil ensemble, potentially statistically significant deviations from random coil behavior are also evident. These small, site-specific deviations may provide a means of reconciling earlier, scattering-based evidence for the random coil nature of the unfolded state with more site-specific spectroscopic evidence suggesting residual structure. We have also studied the unfolded ensemble populated in 50% trifluoroethanol, a denaturant that induces a highly helical unfolded state. We find that the size and shape of the unfolded ensemble under these conditions is effectively indistinguishable from that populated in guanidinium hydrochloride solutions, suggesting that the gross structure of the denatured state is, perhaps surprisingly, independent of the chemistry of the cosolvent.

Escherichia coli Proteins↗

PubMed2004

Exonuclease I hydrolyzes DNA with a distribution of rates.

We report heterogeneity in the time necessary for Exonuclease I to hydrolyze identical DNA fragments. A real-time fluorescence method measured the time required by molecules of Exonuclease I to hydrolyze single-stranded DNA that was synthesized to have two fluorescently labeled nucleotides. One fluorescently labeled nucleotide was located near the 3' end of the DNA and the other near the 5' end. Heterogeneity in the hydrolysis rate of the exonuclease population was inferred from the distribution of times necessary to cleave these DNA fragments. In particular, we found simple first-order kinetics, using a single hydrolysis rate, did not result in a good fit to the data. Better fits to the data were obtained if one assumed a distribution of hydrolysis rates for the exonuclease population. Under our experimental conditions, this broad distribution of rates was centered near 100 nt/s.

DNA↗

PubMed2003

Progress towards single-molecule DNA sequencing: a one color demonstration.

Single molecules of fluorescently labeled nucleotides were detected during the cleavage of individual DNA fragments by a processive exonuclease. In these experiments, multiple (10-100) strands of DNA with tetramethyl rhodamine labeled dUMP (TMR-dUMP) incorporated into the sequence were anchored in flow upstream of the detection region of an ultra sensitive flow cytometer. A dilute solution of Exonuclease I passed over the microspheres. When an exonuclease attached to a strand, processive digestion of that strand began. The liberated, labeled bases flowed through the detection region and were detected at high efficiency at the single-molecule level by laser-induced fluorescence. The digestion of a single strand of DNA by a single exonuclease was discernable in these experiments. This result demonstrates the feasibility of single-molecule DNA sequencing. In addition, these experiments point to a new and practical means of arriving at a consensus sequence by individually reading out identical sequences on multiple fragments.

Color↗

PubMed2003

Fluorescence correlation spectroscopic studies of diffusion within the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate.

Fluorescence correlation spectroscopy (FCS) measurements have been performed in order to determine the translational diffusion coefficients of three differently charged fluorescent probes (cationic: rhodamine 6G, neutral: 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4-H-pyran, DCM, and anionic: fluorescein) dissolved within the common room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, [bmim][PF6]. These experiments demonstrate that FCS is a rapid, sensitive, precise (typical RSD from 4-8%), and low-consumption screening tool for the determination of analyte mobility within microliter ionic liquid samples.

Journal Article↗