Home - Osman Bilsel

Loading …

smFRET/FCS microscope
In order to probe the conformation, dynamics, oligomeric state and kinetics of biological macromolecules we have built a custom single-molecule confocal microscope with time-correlated single-photon capabilities. The ‘scope has a number of features that make it convenient for probing protein-protein interactions:

488 nm picosecond diode laser excitation

594 nm CW laser excitation

idQuantique ultra-low-noise SPAD detectors with up to 4 channel capability (currently 2 are implemented)

Multi-SPC-150 TCSPC electronics

Fully automated
Read More “smFRET/FCS microscope”
CryoEM grid preparation

Recent advances in cryo-electron microscopy (cryoEM), such as the development of direct detectors, automation and 3D particle reconstruction algorithms, have transformed structural biology by enabling investigators to obtain near atomic resolution structures without the need to grow crystals. A significant challenge that limits the wider applicability of cryoEM as a structural tool is the preparation of suitable samples in thin (< 100 nm) layers of vitreous ice in the approximately micron-sized holes of a grid substrate. This arises from the large surface-area-to-volume ratio of the thin aqueous layers prior to freezing of the thin liquid layer in a cryogen. Biological macromolecules preferentially localize to the air-water interface in the thin liquid layer, giving rise to preferred orientations or, in some cases, denaturing.

Read More “CryoEM grid preparation”
Live cell imaging

Live cell microscopy: The other question that we are interested in addressing focuses on understanding the factors leading to toxicity in live cells. To this end. we have built a 2-photon (also capable of 1-photon confocal) laser scanning microscope custom suited for long-time live cell studies. The goal is to track the spatial localization and oligomerization of SOD1 in live cells and correlate that with toxicity.
Small-molecule therapeutic development

If the hypothesis that the gain-of-function toxicity of SOD1 is triggered by a preferential increase in unfolded state population is correct, than one approach to mitigating the destabilization effect of disease-related mutations or zinc deficiency might be to use a small-molecule to preferentially stability the native state.

Read More “Small-molecule therapeutic development”
Single-molecule fluorescence and FCS

Another area that we are interested in is using single-molecule fluorescence methods for studying protein folding reactions, protein-protein interactions and protein oligomer formation. Single-molecule methods can be used to probe protein oligomer formation and also, using fluorescence correlation spectroscopy, probe dynamics from the sub-microsecond to tens of seconds timescale. To enable these studies we have built

Read More “Single-molecule fluorescence and FCS”
Energy landscape of ALS-related proteins

Protein misfolding and aggregation have been implicated in a number of human diseases. One of the proteins that we study, human superoxide dismutase (hSOD1), has been implicated in amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease. As part of a collaborative effort with Jill Zitzewitz and Bob Matthews, we have been studying the

Read More “Energy landscape of ALS-related proteins”
Protein folding

Proteins are known to adopt specific (but dynamic) three-dimensional structures that allow them to carry out their function. It is well recognized that this process doesn’t occur by a random search but is instead a biased search. We are interested in understanding the factors that guide the protein to the correct structure or set of structures.

Read More “Protein folding”

Welcome!

Research

My research focuses on a broad range of topics ranging from protein folding, oligomerization of proteins in neurodegenerative diseases, microfluidic device design and fabrication. I also collaborate with a number of groups (see Collaborations). My research also often involves the development of custom instrumentation. These can be viewed under Instrumentation.

Protein folding

Proteins are known to adopt specific (but dynamic) three-dimensional structures that allow them to carry out their function. It is well recognized that this process doesn’t occur by a random search but is instead a biased search. We are interested in understanding the factors that guide the protein to the correct structure or set of structures.

Energy landscape of ALS-related proteins

Protein misfolding and aggregation have been implicated in a number of human diseases. One of the proteins that we study, human superoxide dismutase (hSOD1), has been implicated in amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease. As part of a collaborative effort with Jill Zitzewitz and Bob Matthews, we have been studying the

Live cell imaging

Live cell microscopy: The other question that we are interested in addressing focuses on understanding the factors leading to toxicity in live cells. To this end. we have built a 2-photon (also capable of 1-photon confocal) laser scanning microscope custom suited for long-time live cell studies. The goal is to track the spatial localization and oligomerization of SOD1 in live cells and correlate that with toxicity.

Single-molecule fluorescence and FCS

Another area that we are interested in is using single-molecule fluorescence methods for studying protein folding reactions, protein-protein interactions and protein oligomer formation. Single-molecule methods can be used to probe protein oligomer formation and also, using fluorescence correlation spectroscopy, probe dynamics from the sub-microsecond to tens of seconds timescale. To enable these studies we have built

Small-molecule therapeutic development

If the hypothesis that the gain-of-function toxicity of SOD1 is triggered by a preferential increase in unfolded state population is correct, than one approach to mitigating the destabilization effect of disease-related mutations or zinc deficiency might be to use a small-molecule to preferentially stability the native state.

Oligomerization mechanism of SOD1

Another part of this study is focused on identifying the small oligomers present and the regions of SOD1 involved in stabilizing these small oligomers. We are pursuing these studies by a number of biophysical tools, including hydrogen exchange mass spec and fast photochemical oxidation (in collaboration with Michael Gross’ lab at Washington University in St. Louis), single-molecule methods and electron microscopy.

Instrumentation

Much of the instrumentation in the lab is custom-built to address a specific question. Below are some of the instruments we have put together. We also typically write our own data acquisition and analysis software.

Time-resolved fluorescence and lifetime-resolved FRET

Time-resolved fluorescence using time-correlated single-photon counting is a versatile tool for biophysical studies. By resolving the nanosecond timescale decay of fluorescence we can obtain information about the local environment of fluorophores (via time-resolved anisotropy, for example) and, with suitable donor and acceptor fluorophores, distance distributions in biological macromolecules. By resolving the lifetime one can also discern sub-populations in heterogeneous samples. For example, a marginally stable protein or peptide may populate both a high-FRET and a low-FRET state. This will be very apparent in the

smFRET/FCS microscope

In order to probe the conformation, dynamics, oligomeric state and kinetics of biological macromolecules we have built a custom single-molecule confocal microscope with time-correlated single-photon capabilities. The ‘scope has a number of features that make it convenient for probing protein-protein interactions:
488 nm picosecond diode laser excitation
594 nm CW laser excitation
idQuantique ultra-low-noise SPAD detectors with up to 4 channel capability (currently 2 are implemented)
Multi-SPC-150 TCSPC electronics
Fully automated
Read More “smFRET/FCS microscope”

Microfluidics and Spectroscopy for Microsecond Folding Kinetics

Protein folding events occur on a wide range of timescales. These are highlighted below with the various techniques used to probe them. One of the limiting factors in protein folding studies has been access to the sub-millisecond timescale for kinetic measurements. Following the lead of the Rousseau, Eaton and Roder groups, we have

CryoEM grid preparation

Recent advances in cryo-electron microscopy (cryoEM), such as the development of direct detectors, automation and 3D particle reconstruction algorithms, have transformed structural biology by enabling investigators to obtain near atomic resolution structures without the need to grow crystals. A significant challenge that limits the wider applicability of cryoEM as a structural tool is the preparation of suitable samples in thin (< 100 nm) layers of vitreous ice in the approximately micron-sized holes of a grid substrate. This arises from the large surface-area-to-volume ratio of the thin aqueous layers prior to freezing of the thin liquid layer in a cryogen. Biological macromolecules preferentially localize to the air-water interface in the thin liquid layer, giving rise to preferred orientations or, in some cases, denaturing.

Autosampler

Overview: Using 3 Zaber stepper motors and a Hamilton Microlab 500 series syringe pump we’ve assembled a home-built autosampler and written custom software for it. The software is written in Labview (originally version 8.2 but currently in Labview 2016) and the software is available here (you’ll need the LabVIEW Runtime Engine to run the executable). We’ve used the unit for automated titrations (urea and guanidineHCl denaturation of proteins) using time-resolved fluorescence and small-angle x-ray scattering (SAXS) and obtained data that would have been very difficult and laborious to obtain manually. Many thanks to Sagar Kathuria for helping