High-Resolution NMR Studies of Human Tissue Factor.

In normal hemostasis, the blood clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to the integral membrane protein, human tissue factor (TF). The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the soluble extracellular domain of TF (sTF) exist; however, these structures are missing electron density in functionally relevant regions of the protein.

Backbone resonance assignments of the C2 domain of coagulation factor VIII.

Factor VIII (FVIII, other clotting factors are named similarly) is a glycoprotein that circulates in the plasma bound to von Willebrand factor. During the blood coagulation cascade, activated FVIII (FVIIIa) binds to FIXa and activates FX in the presence of calcium ions and phospholipid membranes. The C1 and C2 domains mediate membrane binding that is essential for activation of the FVIIIa-FIXa complex.

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers.

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established.

Sublancin is not a lantibiotic but an S-linked glycopeptide.

Sublancin is shown to be an S-linked glycopeptide containing a glucose attached to a cysteine residue, establishing a new post-translational modification. The activity of the S-glycosyl transferase was reconstituted in vitro, and the enzyme is shown to have relaxed substrate specificity, allowing the preparation of analogs of sublancin. Glycosylation is essential for its antimicrobial activity.

Backbone 1H, 13C and 15N resonance assignments of the extracellular domain of tissue factor.

Backbone (1)H, (13)C and (15)N resonance assignments are presented for the extracellular domain of tissue factor. Tissue factor is the integral membrane protein that initiates blood coagulation through the formation an enzymatic complex with the plasma serine protease, factor VIIa.

Protein-phospholipid interactions in blood clotting.

Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations.

Membrane-induced folding of the cAMP-regulated phosphoprotein endosulfine-alpha.

Endosulfine-alpha (ENSA) is a 121-residue cAMP-regulated phosphoprotein, originally identified as an endogenous regulator of ATP-sensitive potassium channels. ENSA has been implicated in the regulation of insulin secretion, and expression of ENSA is decreased in brains of both Alzheimer's disease (AD) and Down's syndrome patients. We recently described membrane-dependent interactions between ENSA and the Parkinson's disease associated protein alpha-synuclein.

Crystal polymorphism of protein GB1 examined by solid-state NMR spectroscopy and X-ray diffraction.

The study of micro- or nanocrystalline proteins by magic-angle spinning (MAS) solid-state NMR (SSNMR) gives atomic-resolution insight into structure in cases when single crystals cannot be obtained for diffraction studies. Subtle differences in the local chemical environment around the protein, including the characteristics of the cosolvent and the buffer, determine whether a protein will form single crystals. The impact of these small changes in formulation is also evident in the SSNMR spectra; however, the changes lead only to correspondingly subtle changes in the spectra.

(1)H, (13)C, and (15)N resonance assignment of the cAMP-regulated phosphoprotein endosulfine-alpha in free and micelle-bound states.

(13)C, (15)N, and (1)H chemical shift assignments are presented for the cAMP-regulated phosphoprotein endosulfine-alpha in its free and micelle-bound states. Secondary chemical shift analysis demonstrates formation of four helices in the micelle-bound state, which are not present in the absence of detergent.

Conformation-specific binding of alpha-synuclein to novel protein partners detected by phage display and NMR spectroscopy.

Alpha-synuclein (AS) is an intrinsically unstructured protein in aqueous solution but is capable of forming beta-sheet-rich fibrils that accumulate as intracytoplasmic inclusions in Parkinson disease and certain other neurological disorders. However, AS binding to phospholipid membranes leads to a distinct change in protein conformation, stabilizing an extended amphipathic alpha-helical domain reminiscent of the exchangeable apolipoproteins. To better understand the significance of this conformational change, we devised a novel bacteriophage display screen to identify protein binding partners of helical AS and have identified 20 proteins with roles in diverse cellular processes related to membrane trafficking, ion channel modulation, redox metabolism, and gene regulation.

Constant-time through-bond 13C correlation spectroscopy for assigning protein resonances with solid-state NMR spectroscopy.

Even as available magnetic fields for NMR continue to increase, resolution remains one of the most critical limitations in assigning and solving structures of larger biomolecules. Here we present a novel constant-time through-bond correlation spectroscopy for solids that offers superior resolution for 13C chemical shift assignments in proteins. In this experiment, the indirect evolution and transfer periods are combined into a single constant time interval, offering increased resolution while not sacrificing sensitivity.