Clay Bracken

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.

Structural Architecture of the CARMA1/Bcl10/MALT1 Signalosome: Nucleation-Induced Filamentous Assembly

The CARMA1/Bcl10/MALT1 (CBM) signalosome mediates antigen receptor-induced NF-κB signaling to regulate multiple lymphocyte functions. While CARMA1 and Bcl10 contain caspase recruitment domains (CARDs), MALT1 is a paracaspase with structural similarity to caspases. Here we show that the reconstituted CBM signalosome is a helical filamentous assembly in which substoichiometric CARMA1 nucleates Bcl10 filaments. Bcl10 filament formation is a highly cooperative process whose threshold is sensitized by oligomerized CARMA1 upon receptor activation. In cells, both cotransfected CARMA1/Bcl10 complex and the endogenous CBM signalosome are filamentous morphologically. Combining crystallography, nuclear magnetic resonance, and electron microscopy, we reveal the structure of the Bcl10 CARD filament and the mode of interaction between CARMA1 and Bcl10. Structure-guided mutagenesis confirmed the observed interfaces in Bcl10 filament assembly and MALT1 activation in vitro and NF-κB activation in cells. These data support a paradigm of nucleation-induced signal transduction with threshold response due to cooperativity and signal amplification by polymerization.

Val66Met polymorphism of BDNF alters prodomain structure to induce neuronal growth cone retraction.

A common single-nucleotide polymorphism in the human brain-derived neurotrophic factor (BDNF) gene results in a Val66Met substitution in the BDNF prodomain region. This single-nucleotide polymorphism is associated with alterations in memory and with enhanced risk to develop depression and anxiety disorders in humans. Here we show that the isolated BDNF prodomain is detected in the hippocampus and that it can be secreted from neurons in an activity-dependent manner. Using nuclear magnetic resonance spectroscopy and circular dichroism we find that the prodomain is intrinsically disordered, and the Val66Met substitution induces structural changes. Surprisingly, application of Met66 (but not Val66) BDNF prodomain induces acute growth cone retraction and a decrease in Rac activity in hippocampal neurons. Expression of p75NTR and differential engagement of the Met66 prodomain to the SorCS2 receptor are required for this effect. These results identify the Met66 prodomain as a new active ligand which modulates neuronal morphology.

Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis

Cardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age‐related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS‐31 (D‐Arg‐dimethylTyr‐Lys‐Phe‐NH2) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS‐31 also protected the electron carrier function of cytochrome c.

NMR spin relaxation methods for characterization of disorder and folding in proteins.

The flexibility and dynamics of proteins directly influence the processes of protein folding, recognition, and function. NMR spin relaxation methods are used to assess the dynamics and mobility of proteins, for fast ps and ns motions as well as slower microsecond and ms events. The degree of protein flexibility and disorder as well as the changes in protein flexibility can be assessed by NMR spin relaxation methods at individual residues within the protein. In addition to probing protein dynamics, the changes in the NMR-derived order parameters can be used to estimate the entropic contributions of order-disorder transitions. Furthermore, kinetic processes in the ms time regime may be directly investigated to extract the rates of conformational interconversion, ligand binding, and protein folding processes. We show how a variety of dynamical information can be obtained from NMR relaxation measurements. We present examples that illustrate the use of NMR spin relaxation analysis for investigation of folding and disorder in proteins.

Sevoflurane binds and allosterically blocks integrin lymphocyte function-associated antigen-1.

Background:Volatile anesthetics have been shown to modify immune cell functions via several mechanisms, some of which have been only partially elucidated. We demonstrated that isoflurane inhibits primary leukocyte integrin lymphocyte function-associated antigen-1 (LFA-1) by binding to the allosteric cavity critical for conformational activation to its high-affinity form. It remains to be determined whether the allosteric inhibition of LFA-1 by isoflurane can be generalized to other anesthetics such as sevoflurane. Methods:The effects of sevoflurane on the ability of LFA-1 to bind to its counter-ligand, intercellular adhesion molecule-1, was studied in leukocytes by flow cytometry. To examine whether sevoflurane acts directly on LFA-1, we measured ligand-binding using beads coated with purified LFA-1 protein. To distinguish between competitive versus allosteric inhibition, we analyzed the effects of sevoflurane on both wild-type and mutant-locked high-affinity LFA-1. One-way analysis of variance was employed for statistical analysis of the data. Nuclear magnetic resonance spectroscopy was used to identify sevoflurane binding site(s). Results:Sevoflurane at clinically relevant concentrations inhibited the ligand-binding function of LFA-1 in leukocytes as well as in cell-free assays (P < 0.05). Sevoflurane blocked wild-type but not locked high-affinity LFA-1, thereby demonstrating an allosteric mode of inhibition. Nuclear magnetic resonance spectroscopy revealed that sevoflurane bound to the allosteric cavity, to which LFA-1 allosteric antagonists and isoflurane also bind. Conclusions:This study suggests that sevoflurane also blocks the activation-dependent conformational changes of LFA-1 to the high-affinity form. The allosteric mode of action exemplified by sevoflurane and isoflurane via LFA-1 might represent one of the underlying mechanisms of anesthetic-mediated immunomodulation.

The volatile anesthetic isoflurane perturbs conformational activation of integrin LFA-1 by binding to the allosteric regulatory cavity

The molecular and structural basis of anesthetic interactions with conformations and functionalities of cell surface receptors remains to be elucidated. We have demonstrated that the widely used volatile anesthetic isoflurane blocks the activation‐dependent conformational conversion of integrin lymphocyte function associated antigen‐1 (LFA‐1), the major leukocyte cell adhesion molecule, to a high‐affinity configuration. Perturbation of LFA‐1 activation by isoflurane at clinically relevant concentrations leads to the inhibition of T‐cell interactions with target cells as well as ligand‐triggered intracellular signaling. Nuclear magnetic resonance spectroscopy reveals that isoflurane binds within a cavity in the LFA‐1 ligand‐binding domain, which is a previously identified drug‐binding site for allosteric small‐molecule antagonists that stabilize LFA‐1 in a low‐affinity conformation. These results provide a potential mechanism for the immunomodulatory properties of isoflurane.— Yuki, K., Astrof, N. S., Bracken, C., Yoo, R., Silkworth, W., Soriano, S. G., Shimaoka, M. The volatile anesthetic isoflurane perturbs conformational activation of integrin LFA‐1 by binding to the allosteric regulatory cavity. FASEB J. 22, 4109–4116 (2008)

Trapping a Folding Intermediate of the α-Helix : Stabilization of the π-Helix

We report the design, synthesis, and characterization of a short peptide trapped in a pi-helix configuration. This high-energy conformation was nucleated by a preorganized pi-turn, which was obtained by replacing an N-terminal intramolecular main chain i and i + 5 hydrogen bond with a carbon-carbon bond. Our studies highlight the nucleation parameter as a key factor contributing to the relative instability of the pi-helix and allow us to estimate fundamental helix-coil transition parameters for this conformation.

Assembling ligands in situ using bioorthogonal boronate ester synthesis

Many molecules that could manipulate cellular function are not practical due to their large size and concomitant undesirable pharmocokinetic properties. Here, we describe a bioorthogonal, highly stable boronate ester (HiSBE) synthesis and use this reaction to synthesize a biologically active molecule from smaller precursors in a physiological context. The rapid rate of HiSBE synthesis suggests that it may be useful for assembling a wide variety of biologically active molecules in physiological solutions.

Helix formation and the unfolded state of a 52‐residue helical protein

A growing class of proteins in biological processes has been found to be unfolded on isolation under normal solution conditions. We have used NMR spectroscopy to characterize the structural and dynamic properties of the unfolded and partially folded states of a 52‐residue alanine‐rich protein (Ala‐14) at temperatures from −5°C to 40°C. At 40°C, alanine residues in Ala‐14 adopt ϕ and ψ angles, consistent with a significant ensemble population of polyproline II conformation. Analysis of relaxation rates in the protein reveals that a series of residues, Gln 35–Ala 36–Ala 37–Lys 38–Asp 39–Asp 40–Ala 41–Ala 42, displays slow motional dynamics at both −5°C and 40°C. Temperature‐dependent chemical shift changes indicate that this region is the site of helix initiation. The remaining N‐terminal residues become increasingly dynamic as they extend from the nucleation site. The C terminus remains dynamic and changes less with temperature, indicating it is relatively unstructured. Ala‐14 provides a high‐resolution portrait of the unfolded state and the process of helix nucleation and propagation in the absence of tertiary contacts, information that bears on early events in protein folding.