Papers by Mariam Iftikhar
Proceedings of SPIE, Feb 10, 2011
ABSTRACT We report first a new derivation of the scattering phase theorem and provide, for the fi... more ABSTRACT We report first a new derivation of the scattering phase theorem and provide, for the first time, the correct relation between the variance of phase gradient and the reduced scattering coefficient. The scattering-phase theorem is then applied to investigate bulk light scattering property from the phase map of thin slices of tissue phantoms measured by a differential phase interference (DIC) microscope using the intensity propagation equation approach. The scattering coefficient, the reduced scattering coefficient, and the anisotropy factor of the sample obtained with this approach is compared to known scattering property of the bulk samples.
ACS Chemical Biology, Mar 20, 2015
Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed... more Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 μs allowed the direct recording of lysozyme's opening and closing transitions. Unexpectedly, both motions required 37 μs, on average. The distribution of transition durations was also independent of the enzyme's state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme's two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 μs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme's state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.
Bulletin of the American Physical Society, Mar 3, 2015
Pushing single molecule techniques to microsecond resolution proves that T4 Lysozyme is a Brownia... more Pushing single molecule techniques to microsecond resolution proves that T4 Lysozyme is a Brownian ratchet MAXIM V. AKHTEROV,
1.2 Importance of enzyme dynamicsinterconversion between states 2 1.3 Techniques that measure tra... more 1.2 Importance of enzyme dynamicsinterconversion between states 2 1.3 Techniques that measure travel time between enzymatic states 4 1.4 SWCNT-FET devices can study microsecond conformational changes 9 1.5 Bioconjugation strategies for device fabrication 10 1.6 References 13 CHAPTER 2: Monitoring the Single-Molecule Dynamics of PKA by 17-54 Functionalization of Single-Walled Carbon Nanotube Circuits 2.1 Abstract 17 2.2 Introduction 18 2.3 Engineering a single cysteine variant of PKA 22 2.4 Experimental setup 28 2.5 Control electronic measurements of PKA 29 2.6 Electronic measurements of PKA binary and ternary complexes 31 2.7 Electrically-driven deposition of peptides onto SWCNTs 39 iv 2.8 PKA and caveolin-1 binding studies 2.9 Conclusions 2.10 Materials and methods 2.11 References CHAPTER 3: Improving the Cysteine-Maleimide Bioconjugation 55-81 for SWCNT-FETs
Langmuir, Oct 4, 2012
Cytochrome c (cyt. c) has been encapsulated in silica sol-gels and processed to form bioaerogels ... more Cytochrome c (cyt. c) has been encapsulated in silica sol-gels and processed to form bioaerogels with gas-phase activity for nitric oxide through a simplified synthetic procedure. Previous reports demonstrated a need to adsorb cyt. c to metal nanoparticles prior to silica sol-gel encapsulation and processing to form aerogels. We report that cyt. c can be encapsulated in aerogels without added nanoparticles and retain structural stability and gas-phase activity for nitric oxide. While the UV-visible Soret absorbance and nitric oxide response indicate that cyt. c encapsulated with nanoparticles in aerogels remains slightly more stable and functional than cyt. c encapsulated alone, these properties are not very different in the two types of aerogels. From UV-visible and Soret circular dichroism results, we infer that cyt. c encapsulated alone self-organizes to reduce contact with the silica gel in a way that may bear at least some resemblance to the way cyt. c self-organizes into superstructures of protein within aerogels when nanoparticles are present. Both the buffer concentration and the cyt. c concentration of solutions used to synthesize the bioaerogels affect the structural integrity of the protein encapsulated alone within the dried aerogels. Optimized bioaerogels are formed when cyt. c is encapsulated from 40 mM phosphate buffered solutions, and when the loaded cyt. c concentration in the aerogel is in the range of 5 to 15 μM. Increased viability of cyt. c in aerogels is also observed when supercritical fluid used to produce aerogels is vented over relatively long times.
International Journal of Molecular Sciences
Understanding complex biological events at the molecular level paves the path to determine mechan... more Understanding complex biological events at the molecular level paves the path to determine mechanistic processes across the timescale necessary for breakthrough discoveries. While various conventional biophysical methods provide some information for understanding biological systems, they often lack a complete picture of the molecular-level details of such dynamic processes. Studies at the single-molecule level have emerged to provide crucial missing links to understanding complex and dynamic pathways in biological systems, which are often superseded by bulk biophysical and biochemical studies. Latest developments in techniques combining single-molecule manipulation tools such as optical tweezers and visualization tools such as fluorescence or label-free microscopy have enabled the investigation of complex and dynamic biomolecular interactions at the single-molecule level. In this review, we present recent advances using correlated single-molecule manipulation and visualization-based...
Author(s): Iftikhar, Mariam | Advisor(s): Weiss, Gregory A | Abstract: Molecular motions of prote... more Author(s): Iftikhar, Mariam | Advisor(s): Weiss, Gregory A | Abstract: Molecular motions of proteins and their flexibility induce conformational states required for enzyme catalysis, signal transduction, and protein-protein interactions. However, the mechanisms for protein transitions between conformational states are often poorly understood, especially in the millisecond to microsecond range where conventional optical techniques and computational modeling are most limited. To investigate the microsecond dynamics of enzymes, single-walled carbon nanotube – field-effect transistors (SWCNT-FETs) were used as single-molecule biosensors. The SWCNT-FETs have sufficient sensitivity and bandwidth to monitor the conformational motions and processivity of an individual cAMP-dependent protein kinase A (PKA) molecule. Protein attachment is accomplished by functionalizing a SWCNT-FET device with a single protein and measuring the conductance versus time through the device as it is submerged in ...
Biochimica et biophysica acta, Jan 29, 2018
Protein engineering by directed evolution can alter proteins' structures, properties, and fun... more Protein engineering by directed evolution can alter proteins' structures, properties, and functions. However, membrane proteins, despite their importance to living organisms, remain relatively unexplored as targets for protein engineering and directed evolution. This gap in capabilities likely results from the tendency of membrane proteins to aggregate and fail to overexpress in bacteria cells. For example, the membrane protein caveolin-1 has been implicated in many cell signaling pathways and diseases, yet the full-length protein is too aggregation-prone for detailed mutagenesis, directed evolution, and biophysical characterization. Using a phage-displayed library of full-length caveolin-1 variants, directed evolution with alternating subtractive and functional selections isolated a full-length, soluble variant, termed cav, for expression in E. coli. Cav folds correctly and binds to its known protein ligands HIV gp41, the catalytic domain of cAMP-dependent protein kinase A, and...
2013 Transducers & Eurosensors XXVII: The 17th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS & EUROSENSORS XXVII), 2013
For over a decade, researchers have pursued sensitive, label-free biosensing and biodetection usi... more For over a decade, researchers have pursued sensitive, label-free biosensing and biodetection using nanoscale electronic devices like field-effect transistors. Prototype devices made with single-walled carbon nanotubes proved to have enough sensitivity to detect single molecule events, as well as sufficient bandwidth to monitor real time dynamics. Combining these characteristics has led to novel electronic measurements where the bond-by-bond chemical activity of single enzyme molecules is monitored in real time. The high bandwidth of the nanotube transistors allow every individual chemical event to be clearly resolved, providing excellent statistics from tens of thousands of turnovers by a single molecule.
Langmuir, 2012
Cytochrome c (cyt. c) has been encapsulated in silica sol-gels and processed to form bioaerogels ... more Cytochrome c (cyt. c) has been encapsulated in silica sol-gels and processed to form bioaerogels with gas-phase activity for nitric oxide through a simplified synthetic procedure. Previous reports demonstrated a need to adsorb cyt. c to metal nanoparticles prior to silica sol-gel encapsulation and processing to form aerogels. We report that cyt. c can be encapsulated in aerogels without added nanoparticles and retain structural stability and gas-phase activity for nitric oxide. While the UV-visible Soret absorbance and nitric oxide response indicate that cyt. c encapsulated with nanoparticles in aerogels remains slightly more stable and functional than cyt. c encapsulated alone, these properties are not very different in the two types of aerogels. From UV-visible and Soret circular dichroism results, we infer that cyt. c encapsulated alone self-organizes to reduce contact with the silica gel in a way that may bear at least some resemblance to the way cyt. c self-organizes into superstructures of protein within aerogels when nanoparticles are present. Both the buffer concentration and the cyt. c concentration of solutions used to synthesize the bioaerogels affect the structural integrity of the protein encapsulated alone within the dried aerogels. Optimized bioaerogels are formed when cyt. c is encapsulated from 40 mM phosphate buffered solutions, and when the loaded cyt. c concentration in the aerogel is in the range of 5 to 15 μM. Increased viability of cyt. c in aerogels is also observed when supercritical fluid used to produce aerogels is vented over relatively long times.
Pushing single molecule techniques to microsecond resolution proves that T4 Lysozyme is a Brownia... more Pushing single molecule techniques to microsecond resolution proves that T4 Lysozyme is a Brownian ratchet MAXIM V. AKHTEROV,
ABSTRACT Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity an... more ABSTRACT Using single-walled carbon nanotube (SWNT) transistors, we monitored the processivity and dynamics of single molecules of cAMP-dependent protein kinase (PKA). As PKA enzymatically phosphorylates its peptide substrate, it generates an electronic signal in the transistor that can be monitored continuously and with 20 μs resolution. The electronic recording directly resolves substrate binding, ATP binding, and cooperative formation of PKA&#39;s catalytically functional, ternary complex. Statistical analysis of many events determines on- and off-rates for each of these events, as well as the full transistion probability matrix between them. Long duration monitoring further revealed minute-to-minute rate variability for a single molecule, and different mechanistic statistics for ATP binding than for substrate. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute that is useful for an enzyme that plays crucial roles in cell signaling.
Sol-gel-based aerogels are three-dimensional, nanoscale materials that combine large surface area... more Sol-gel-based aerogels are three-dimensional, nanoscale materials that combine large surface areas and high porosities. These traits make them useful for sensing or electrochemical applications when chemical dopants are incorporated into the gels to add functionality. Cytochrome c (cyt. c) has been successfully doped into aerogels through the incorporation of gold (or silver) nanoparticle nucleated protein superstructures. There is potential to make use of these biomolecular aerogels in bioanalytical devices. We have chosen to explore the electrochemical properties of Au~cyt. c superstructures in buffered media, outside of the aerogel nanoarchitecture, in order to further our understanding of these stabilizing superstructures. We will present how the electron-transfer rate constant, formal potential, electrode surface coverage, and electrochemical reversibility of cyt. c within a gold nanoparticle-stabilized superstructure compares to that of unassociated cyt. c. Additionally, our r...
Biophysical Journal, 2014
ACS chemical biology, Jan 20, 2015
Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed... more Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 μs allowed the direct recording of lysozyme's opening and closing transitions. Unexpectedly, both motions required 37 μs, on average. The distribution of transition durations was also independent of the enzyme's state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme's two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysoz...
ACS Chemical Biology, 2015
Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed... more Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 μs allowed the direct recording of lysozyme&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s opening and closing transitions. Unexpectedly, both motions required 37 μs, on average. The distribution of transition durations was also independent of the enzyme&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 μs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.
Optical Tomography and Spectroscopy of Tissue IX, 2011
ABSTRACT We report first a new derivation of the scattering phase theorem and provide, for the fi... more ABSTRACT We report first a new derivation of the scattering phase theorem and provide, for the first time, the correct relation between the variance of phase gradient and the reduced scattering coefficient. The scattering-phase theorem is then applied to investigate bulk light scattering property from the phase map of thin slices of tissue phantoms measured by a differential phase interference (DIC) microscope using the intensity propagation equation approach. The scattering coefficient, the reduced scattering coefficient, and the anisotropy factor of the sample obtained with this approach is compared to known scattering property of the bulk samples.
Biophysical Journal, 2015
Carbon Nanotubes, Graphene, and Associated Devices VI, 2013
ABSTRACT Nanoscale electronic devices like field-effect transistors have long promised to provide... more ABSTRACT Nanoscale electronic devices like field-effect transistors have long promised to provide sensitive, label-free detection of biomolecules. In particular, single-walled carbon nanotubes have the requisite sensitivity to detect single molecule events and sufficient bandwidth to directly monitor single molecule dynamics in real time. Recent measurements have demonstrated this premise by monitoring the dynamic, single-molecule processivity of three different enzymes: lysozyme, protein Kinase A, and the Klenow fragment of DNA polymerase I. In each case, recordings resolved detailed trajectories of tens of thousands of individual chemical events and provided excellent statistics for single-molecule events. This electronic technique has a temporal resolution approaching 1 microsecond, which provides a new window for observing brief, intermediate transition states. In addition, the devices are indefinitely stable, so that the same molecule can be observed for minutes and hours. The extended recordings provide new insights into rare events like transitions to chemically-inactive conformations.
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Papers by Mariam Iftikhar