Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D ²H–¹³C–¹³C Solid-State NMR Spectroscopy

²H quadrupolar line shapes deliver rich information about protein dynamics. A newly designed 3D ²H–¹³C–¹³C solid-state NMR magic angle spinning (MAS) experiment is presented and demonstrated on the microcrystalline β1 immunoglobulin binding domain of protein G (GB1). The implementation of ²H–¹³C adiabatic rotor-echo-short-pulse-irradiation cross-polarization (RESPIRATION CP) ensures the accuracy of the extracted line shapes and provides enhanced sensitivity relative to conventional CP methods. The 3D ²H–¹³C–¹³C spectrum reveals ²H line shapes for 140 resolved aliphatic deuterium sites. Motional-averaged ²H quadrupolar parameters obtained from the line-shape fitting identify side-chain motions. Restricted side-chain dynamics are observed for a number of polar residues including K13, D22, E27, K31, D36, N37, D46, D47, K50, and E56, which we attribute to the effects of salt bridges and hydrogen bonds. In contrast, we observe significantly enhanced side-chain flexibility for Q2, K4, K10, E15, E19, N35, N40, and E42, due to solvent exposure and low packing density. T11, T16, and T17 side chains exhibit motions with larger amplitudes than other Thr residues due to solvent interactions. The side chains of L5, V54, and V29 are highly rigid because they are packed in the core of the protein. High correlations were demonstrated between GB1 side-chain dynamics and its biological function. Large-amplitude side-chain motions are observed for regions contacting and interacting with immunoglobulin G (IgG). In contrast, rigid side chains are primarily found for residues in the structural core of the protein that are absent from protein binding and interactions

¹H‑Detected REDOR with Fast Magic-Angle Spinning of a Deuterated Protein

Rotational echo double resonance (REDOR) is a highly successful method for heteronuclear distance determination in biological solid-state NMR, and ¹H detection methods have emerged in recent years as a powerful approach to improving sensitivity and resolution for small sample quantities by utilizing fast magic-angle spinning (>30 kHz) and deuteration strategies. In theory, involving ¹H as one of the spins for measuring REDOR effects can greatly increase the distance measurement range, but few experiments of this type have been reported. Here we introduce a pulse sequence that combines frequency-selective REDOR (FSR) with ¹H detection. We demonstrate this method with applications to samples of uniformly ¹³C,¹⁵N,²H-labeled alanine and uniformly ¹³C,²H,¹⁵N-labeled GB1 protein, back-exchanged with 30% H₂O and 70% D₂O, employing a variety of frequency-selective ¹³C pulses to highlight unique spectral features. The resulting, robust REDOR effects provide (1) tools for resonance assignment, (2) restraints of secondary structure, (3) probes of tertiary structure, and (4) approaches to determine the preferred orientation of aromatic rings in the protein core

Tissue Factor Residues That Modulate Magnesium-Dependent Rate Enhancements of the Tissue Factor/Factor VIIa Complex

The blood coagulation cascade is initiated when the cell-surface complex of factor VIIa (FVIIa, a trypsin-like serine protease) and tissue factor (TF, an integral membrane protein) proteolytically activates factor X (FX). Both FVIIa and FX bind to membranes via their γ-carboxyglutamate-rich domains (GLA domains). GLA domains contain seven to nine bound Ca²⁺ ions that are critical for their folding and function, and most biochemical studies of blood clotting have employed supraphysiologic Ca²⁺ concentrations to ensure saturation of these domains with bound Ca²⁺. Recently, it has become clear that, at plasma concentrations of metal ions, Mg²⁺ actually occupies two or three of the divalent metal ion-binding sites in GLA domains, and that these bound Mg²⁺ ions are required for full function of these clotting proteins. In this study, we investigated how Mg²⁺ influences FVIIa enzymatic activity. We found that the presence of TF was required for Mg²⁺ to enhance the rate of FX activation by FVIIa, and we used alanine-scanning mutagenesis to identify TF residues important for mediating this response to Mg²⁺. Several TF mutations, including those at residues G164, K166, and Y185, blunted the ability of Mg²⁺ to enhance the activity of the TF/FVIIa complex. Our results suggest that these TF residues interact with the GLA domain of FX in a Mg²⁺-dependent manner (although effects of Mg²⁺ on the FVIIa GLA domain cannot be ruled out). Notably, these TF residues are located within or immediately adjacent to the putative substrate-binding exosite of TF

NMR Determination of Protein p<i>K</i>_a Values in the Solid State

Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the p<i>K</i>_a values of large, membrane, or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble β1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly ¹³C,¹⁵N-labeled protein were monitored over several samples varying in pH; p<i>K</i>_a values were determined from these shifts for E27, D36, and E42, and the bounds for the p<i>K</i>_a of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated p<i>K</i>_a values give insights into the crystal packing of the protein

Structural Intermediates during α-Synuclein Fibrillogenesis on Phospholipid Vesicles

α-Synuclein (AS) fibrils are the main protein component of Lewy bodies, the pathological hallmark of Parkinson’s disease and other related disorders. AS forms helices that bind phospholipid membranes with high affinity, but no atomic level data for AS aggregation in the presence of lipids is yet available. Here, we present direct evidence of a conversion from α-helical conformation to β-sheet fibrils in the presence of anionic phospholipid vesicles and direct conversion to β-sheet fibrils in their absence. We have trapped intermediate states throughout the fibril formation pathways to examine the structural changes using solid-state NMR spectroscopy and electron microscopy. The comparison between mature AS fibrils formed in aqueous buffer and those derived in the presence of anionic phospholipids demonstrates no major changes in the overall fibril fold. However, a site-specific comparison of these fibrillar states demonstrates major perturbations in the <i>N</i>-terminal domain with a partial disruption of the long β-strand located in the 40s and small perturbations in residues located in the “non-β amyloid component” (NAC) domain. Combining all these results, we propose a model for AS fibrillogenesis in the presence of phospholipid vesicles

High-Resolution NMR Studies of Human Tissue Factor

<div><p>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. In this context, NMR can provide complementary structural information as well as dynamic insights into enzyme activity. The resolution and sensitivity for NMR studies are greatly enhanced by the ability to prepare multiple milligrams of protein with various isotopic labeling patterns. Here, we demonstrate high-yield production of several isotopically labeled forms of recombinant sTF, allowing for high-resolution NMR studies both in the solid and solution state. We also report solution NMR spectra at sub-mM concentrations of sTF, ensuring the presence of dispersed monomer, as well as the first solid-state NMR spectra of sTF. Our improved sample preparation and precipitation conditions have enabled the acquisition of multidimensional NMR data sets for TF chemical shift assignment and provide a benchmark for TF structure elucidation.</p></div

Components of media used for isotopically labeled growths of sTF.

<p>Components of media used for isotopically labeled growths of sTF.</p

SDS-PAGE analysis of sTF purification.

<p>SDS-PAGE 12% acrylamide gel of sTF samples stained with Coomassie Brilliant Blue R-250 showing: Lane 1: Precision Plus Protein^TM Dual Color marker (Bio-Rad, Hercules, CA, USA); Lane 2: water and sucrose combined supernatant; Lane 3: Q-Sepharose® Fast Flow supernatant; Lane 4: Post 0.45 μm filter; Lane 5: pre-load on Ni²⁺ affinity column; Lane 6: 500 mM imidazole elution; Lane 7: concentrated sTF.</p

Solid-state ¹⁵N-¹³CA correlation spectra of uniform and glycerol labeled sTF.

<p>(A) ¹⁵N-¹³CA correlation spectrum of ~30 mg of uniform ¹³C,¹⁵N ammonium sulfate-precipitated sTF with 5 mM Cu-EDTA. The spectrum was acquired for 3 hours and 20 minutes with an acquisition time of 25 ms. (B) ¹⁵N-¹³CA correlation spectrum of ~25 mg of 2-¹³C glycerol, ²H, ¹⁵N ammonium sulfate-precipitated sTF with 5 mM Cu-EDTA. The spectrum was acquired for 3 hours with an acquisition time of 30 ms. Both spectra were collected at 750 MHz (¹H frequency) at a variable temperature set point of -5°C with a MAS rate of 12.5 kHz. Both spectra were processed with 10 Hz of Lorentzian-to-Gaussian line broadening in each dimension, sine bell apodization, and zero filled 8192 points in the direct (F1) dimension and 2048 in the indirect (F2) dimension with contours drawn at 6 times the noise floor. (C) ¹³C 1D slices extracted from the ¹⁵N-¹³CA 2D spectrum of uniform ¹³C-¹⁵N ammonium sulfate-precipitated sTF shown in A. The corresponding ¹⁵N chemical shifts in the indirect dimension are indicated. (D) ¹³C 1D slices extracted from the ¹⁵N-¹³CA 2D spectrum of 2-¹³C glycerol, ²H, ¹⁵N ammonium sulfate-precipitated sTF shown in B. The corresponding ¹⁵N chemical shifts in the indirect dimension are indicated.</p

¹H-¹⁵N 2D HSQC correlation spectrum.

<p>Solution NMR fingerprint spectrum of 100 μM uniform ¹³C,¹⁵N sTF in 50 mM sodium phosphate (pH 6.5), 50 mM NaCl, 5% DSS, 10% D₂O, 0.1% NaN₃ acquired at a variable temperature set point of 35°C on a Varian/Agilent VNMRS 17.6 T (750 MHz ¹H frequency) spectrometer.</p