21 research outputs found
Using nanoscale bioreactors to characterize sub-populations of CHO clones and screen transfected pools
Traditional means to quantify growth and production rates for antibody-expressing CHO lines involve sampling aliquots and supernatants from well plates that have been seeded with single cells. The number of clones studied is often limited by cloning efficiencies (typically 5-50%) and the inability to handle large numbers of well plates. The speed at which each clone can be measured is limited by the growth rates of cells and the number of cells required to perform each assay. Both of these factors lead to a practical throughput of 100s of clones screened over the course of 2-4 weeks. Furthermore, each readout of a clone offers little to no insight into the behavior of sub-populations within each clone since the aliquot or supernatant is just a small sample representing the entire population.
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Single Molecule Conformational Memory Extraction: P5ab RNA Hairpin
Extracting kinetic models from single
molecule data is an important
route to mechanistic insight in biophysics, chemistry, and biology.
Data collected from force spectroscopy can probe discrete hops of
a single molecule between different conformational states. Model extraction
from such data is a challenging inverse problem because single molecule
data are noisy and rich in structure. Standard modeling methods normally
assume (i) a prespecified number of discrete states and (ii) that
transitions between states are Markovian. The data set is then fit
to this predetermined model to find a handful of rates describing
the transitions between states. We show that it is unnecessary to
assume either (i) or (ii) and focus our analysis on the zipping/unzipping
transitions of an RNA hairpin. The key is in starting with a very
broad class of non-Markov models in order to let the data guide us
toward the best model from this very broad class. Our method suggests
that there exists a folding intermediate for the P5ab RNA hairpin
whose zipping/unzipping is monitored by force spectroscopy experiments.
This intermediate would not have been resolved if a Markov model had
been assumed from the onset. We compare the merits of our method with
those of others
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An investigation of the mechanical properties of the molten globule state of apomyoglobin
Single molecule force spectroscopy has provided important insights into the properties and mechanisms of protein folding. However, there are still many unanswered questions about how force affects the folding and unfolding of proteins and, in particular, the relationship between force and the rate-limiting transition state. In this thesis, I developed two protein systems to address two specific questions. The first question arose form previous work on E. coli RNAse H, in which a molten globule-like intermediate was observed to have a large distance (5 ± 1 nm) to the transition state. This large distance was in sharp contrast to the smaller distances (< 2 nm) typically observed for natively folded proteins. This raised the question of whether this distance was a general property of the E. coli RNAse H intermediate or a more general property of a molten globule state. To this end, I investigated the equilibrium molten globule state of sperm whale apomyoglobin at pH 5 under force and demonstrated that this state had a large distance to the transition state of 6.1 ± 0.5 nm. Further, this state was shown to have a large distance to the transition state regardless of the axis of the applied force. This work suggests that a large distance to the transition state is a general property of the molten globule state. The second system was developed using the SH3 domain from chicken c-Src in order to investigate if and how the structure of the transition state changes under force. I investigated the behavior of the protein under two different force axes observing significant differences in the mechanical unfolding of the protein. These experiments are ongoing but indicate that the change in behavior is because of a change in the structure of the transition state under force. Finally, investigating the properties of the molten globule state revealed an error in previous methodology using constant force feedback experiments. In this thesis, I identify and explain the origin of this error. Further, work on the molten globule state required higher fidelity data and a more sophisticated approach for the analysis of the data. Working with John Chodera and colleagues, we implemented novel methods for the analysis of the data
Recommended from our members
An investigation of the mechanical properties of the molten globule state of apomyoglobin
Single molecule force spectroscopy has provided important insights into the properties and mechanisms of protein folding. However, there are still many unanswered questions about how force affects the folding and unfolding of proteins and, in particular, the relationship between force and the rate-limiting transition state. In this thesis, I developed two protein systems to address two specific questions. The first question arose form previous work on E. coli RNAse H, in which a molten globule-like intermediate was observed to have a large distance (5 ± 1 nm) to the transition state. This large distance was in sharp contrast to the smaller distances (< 2 nm) typically observed for natively folded proteins. This raised the question of whether this distance was a general property of the E. coli RNAse H intermediate or a more general property of a molten globule state. To this end, I investigated the equilibrium molten globule state of sperm whale apomyoglobin at pH 5 under force and demonstrated that this state had a large distance to the transition state of 6.1 ± 0.5 nm. Further, this state was shown to have a large distance to the transition state regardless of the axis of the applied force. This work suggests that a large distance to the transition state is a general property of the molten globule state. The second system was developed using the SH3 domain from chicken c-Src in order to investigate if and how the structure of the transition state changes under force. I investigated the behavior of the protein under two different force axes observing significant differences in the mechanical unfolding of the protein. These experiments are ongoing but indicate that the change in behavior is because of a change in the structure of the transition state under force. Finally, investigating the properties of the molten globule state revealed an error in previous methodology using constant force feedback experiments. In this thesis, I identify and explain the origin of this error. Further, work on the molten globule state required higher fidelity data and a more sophisticated approach for the analysis of the data. Working with John Chodera and colleagues, we implemented novel methods for the analysis of the data
Development of a Protein Chip:Â A MS-Based Method for Quantitation of Protein Expression and Modification Levels Using an Immunoaffinity Approach
Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase
The replication-dependent histone mRNAs, the only eukaryotic mRNAs that do not have poly(A) tails, are present only in S-phase cells. Coordinate posttranscriptional regulation of histone mRNAs is mediated by the stem-loop at the 3′ end of histone mRNAs. The protein that binds the 3′ end of histone mRNA, stem-loop binding protein (SLBP), is required for histone pre-mRNA processing and is involved in multiple aspects of histone mRNA metabolism. SLBP is also regulated during the cell cycle, accumulating as cells enter S phase and being rapidly degraded as cells exit S phase. Mutation of any residues in a TTP sequence (amino acids 60 to 62) or mutation of a consensus cyclin binding site (amino acids 99 to 104) stabilizes SLBP in G(2) and mitosis. These two threonines are phosphorylated in late S phase, as determined by mass spectrometry (MS) of purified SLBP from late S-phase cells, triggering SLBP degradation. Cells that express a stable SLBP still degrade histone mRNA at the end of S phase, demonstrating that degradation of SLBP is not required for histone mRNA degradation. Nuclear extracts from G(1) and G(2) cells are deficient in histone pre-mRNA processing, which is restored by addition of recombinant SLBP, indicating that SLBP is the only cell cycle-regulated factor required for histone pre-mRNA processing