Andrew L. Lee

Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.

Hidden dynamic allostery in a PDZ domain

Structure–function relationships in proteins are predicated on the spatial proximity of noncovalently interacting groups of atoms. Thus, structural elements located away from a proteins active site are typically presumed to serve a stabilizing or scaffolding role for the larger structure. Here we report a functional role for a distal structural element in a PDZ domain, even though it is not required to maintain PDZ structure. The third PDZ domain from PSD-95/SAP90 (PDZ3) has an unusual additional third alpha helix (α3) that packs in contiguous fashion against the globular domain. Although α3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of α3 reduces ligand affinity by 21-fold. Further investigation revealed that the difference in binding free energies between the full-length and truncated constructs is predominantly entropic in nature and that without α3, picosecond-nanosecond side-chain dynamics are enhanced throughout the domain, as determined by 2H methyl NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of α3 and characterization of side-chain dynamics, this dynamic allostery would have gone unnoticed. Moreover, what appeared at first to be an artificial modification of PDZ3 has been corroborated by experimentally verified phosphorylation of α3, revealing a tangible biological mechanism for this novel regulatory scheme. This hidden dynamic allostery raises the possibility of as-yet unidentified or untapped allosteric regulation in this PDZ domain and is a very clear example of function arising from dynamics rather than from structure.

Using NMR to study fast dynamics in proteins: methods and applications

Proteins exist not as singular structures with precise coordinates, but rather as fluctuating bodies that move rapidly through an enormous number of conformational substates. These dynamics have important implications for understanding protein function and for structure-based drug design. NMR spectroscopy is particularly well suited to characterize the dynamics of proteins and other molecules in solution at atomic resolution. Here, NMR relaxation methods for characterizing thermal motions on the picosecond-nanosecond (ps-ns) timescale are reviewed. Motion on this timescale can be conveniently captured by the Lipari-Szabo order parameter, S², a bond-specific measure of restriction of motion. Approaches for determining order parameters are discussed, as are recent examples from the literature that link ps-ns dynamics with conformational entropy, allostery, and protein function in general.

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes, and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times, or koff, should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into koff for a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase that vary by six orders of magnitude in binding affinity. NMR relaxation dispersion experiments revealed a common set of residues near the binding site that undergo a concerted, millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with Ki and koff, suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although kconf,forward is faster than koff, use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.

Phosphorylation of a PDZ domain extension modulates binding affinity and interdomain interactions in postsynaptic density-95 (PSD-95) protein, a membrane-associated guanylate kinase (MAGUK).

Postsynaptic density-95 is a multidomain scaffolding protein that recruits glutamate receptors to postsynaptic sites and facilitates signal processing and connection to the cytoskeleton. It is the leading member of the membrane-associated guanylate kinase family of proteins, which are defined by the PSD-95/Discs large/ZO-1 (PDZ)-Src homology 3 (SH3)-guanylate kinase domain sequence. We used NMR to show that phosphorylation of conserved tyrosine 397, which occurs in vivo and is located in an atypical helical extension (α3), initiates a rapid equilibrium of docked and undocked conformations. Undocking reduced ligand binding affinity allosterically and weakened the interaction of PDZ3 with SH3 even though these domains are separated by a ∼25-residue linker. Additional phosphorylation at two linker sites further disrupted the interaction, implicating α3 and the linker in tuning interdomain communication. These experiments revealed a novel mode of regulation by a detachable PDZ element and offer a first glimpse at the dynamic interaction of PDZ and SH3-guanylate kinase domains in membrane-associated guanylate kinases.

Conservation of Side-Chain Dynamics Within a Protein Family

The question of protein dynamics and its relevance to function is currently a topic of great interest. Proteins are particularly dynamic at the side-chain level on the time scale of picoseconds to nanoseconds. Here, we present a comparison of NMR-monitored side-chain motion between three PDZ domains of approximately 30% sequence identity and show that the side-chain dynamics display nontrivial conservation. Methyl (2)H relaxation was carried out to determine side-chain order parameters (S(2)), which were found to be more similar than naively expected from sequence, local packing, or a combination of the two. Thus, the dynamics of a rather distant homologue appears to be an excellent predictor of a proteins side-chain dynamics and, on average, better than current structure-based methods. Fast side-chain dynamics therefore display a high level of organization associated with global fold. Beyond simple conservation, the analysis herein suggests that the pattern of side-chain flexibility has significant contributions from nonlocal elements of the PDZ fold, such as correlated motions, and that the conserved dynamics may directly support function.

Imaging of STAT3 Signaling Pathway During Mouse Embryonic Stem Cell Differentiation

Signal transducers and activators of transcription 3 (STAT3) is a pleiotropic transcription factor involved in a variety of physiological processes. STAT3 acts as a key transcriptional determinant of mouse embryonic stem (ES) cell self-renewal and plays a pivotal function in early mammalian embryogenesis because the development of many organs requires STAT3 activation. However, little is known about the role of STAT3 function during ES cell differentiation. To answer this question, we built a lentiviral construct with 7-repeat STAT3-binding sequence (enhancer) and minimal TA (promoter) driving renilla luciferase and monomeric red fluorescence protein (Rluc-mRFP), followed by a constitutive cytomegalovirus promoter driving green fluorescent protein as a selection marker. The specificity of our custom-designed 7-repeat STAT3 reporter construct was first confirmed by cotransfection with constitutively active version of STAT3 (STAT3C) into human embryonic kidney 293T cells. Next, a mouse ES cell line stably transduced with STAT3 reporter construct was isolated. This ES cell line showed a tight response in reporter gene expression with leukemia inhibitory factor (LIF) induction and was chosen as a developmental model for the STAT3 functional study. Using serial noninvasive bioluminescence imaging, we showed that the onset of embryoid body (EB) formation involved inhibition of STAT3 activity. However, during differentiation, STAT3 activity steadily increased from day 5 to 14 and was reduced by day 21. STAT3 activity was also confirmed separately by Western blots. Finally, phosphorylation of STAT3 was also found to correspond with cardiomyocyte differentiation. In summary, this is the first study to monitor real-time STAT3 activity during ES cell differentiation. This genetically modified line can be used to study the biological role of STAT3 during ES cell differentiation into different derivatives.

Pathway for unfolding of ubiquitin in sodium dodecyl sulfate, studied by capillary electrophoresis.

This paper characterizes the complexes formed by a small protein, ubiquitin (UBI), and a negatively charged surfactant, sodium dodecyl sulfate (SDS), using capillary electrophoresis (CE), circular dichroism (CD), and amide hydrogen-deuterium exchange (HDX; as monitored by mass spectroscopy, MS). Capillary electrophoresis of complexes of UBI and SDS, at apparent equilibrium, at concentrations of SDS ranging from sub-micellar and sub-denaturing to micellar and denaturing, revealed multiple complexes of UBI and SDS of the general composition UBI-SDS(n). Examination of electrophoretic mobilities of complexes of UBI and SDS as a function of the concentration of SDS provided a new way to characterize the interaction of this protein with SDS and established key characteristics of this system: e.g., the reversibility of the formation of the complexes, their approximate chemical compositions, and the pathway of SDS binding to UBI. The work identified, in addition to SDS-saturated UBI, at least six groups of complexes of UBI with SDS, within which four groups were populated with complexes of distinct stoichiometries: UBI-SDS(approximately 11), UBI-SDS(approximately 25), UBI-SDS(approximately 33), and UBI-SDS(approximately 42). CD spectroscopy and amide HDX of the UBI-SDS(n) complexes suggested that many of the UBI-SDS(n) complexes (n > 11) have greater alpha-helical content than native UBI. Capillary electrophoresis provides a level of detail about interactions of proteins and SDS that has not previously been accessible, and CE is an analytical and biophysical method for studies of interactions of proteins and surfactants that is both convenient and practical. This study sheds light on the formation of the enigmatic protein-SDS complexes formed during SDS polyacrylamide gel electrophoresis and brings a new tool to the study of proteins and detergents.

Segmental Motions, Not a Two-State Concerted Switch, Underlie Allostery in CheY

The switch between an inactive and active conformation is an important transition for signaling proteins, yet the mechanisms underlying such switches are not clearly understood. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. By using 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the μs-ms timescale motions underlying CheY allostery are segmental in nature. We propose an expanded allosteric network of residues, including W58, that undergo asynchronous, local switching between inactive and active-like conformations as the primary basis for the allosteric mechanism.

Infochemistry and infofuses for the chemical storage and transmission of coded information

This article describes a self-powered system that uses chemical reactions—the thermal excitation of alkali metals—to transmit coded alphanumeric information. The transmitter (an “infofuse”) is a strip of the flammable polymer nitrocellulose patterned with alkali metal ions; this pattern encodes the information. The wavelengths of 2 consecutive pulses of light represent each alphanumeric character. While burning, infofuses transmit a sequence of pulses (at 5–20 Hz) of atomic emission that correspond to the sequence of metallic salts (and therefore to the encoded information). This system combines information technology and chemical reactions into a new area—“infochemistry”—that is the first step toward systems that combine sensing and transduction of chemical signals with multicolor transmission of alphanumeric information.