HLA topography enforces shared and convergent immunodominant B cell and antibody alloresponses in transplant recipients.

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. The rules governing the immunogenicity of non-self donor HLA are poorly understood. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific B cell response in the kidney and blood of a transplant recipient with antibody-mediated rejection (AMR).

A biradical-tagged phospholipid as a polarizing agent for solid-state MAS Dynamic Nuclear Polarization NMR of membrane proteins.

A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix.

Solid-State NMR Provides Evidence for Small-Amplitude Slow Domain Motions in a Multispanning Transmembrane α-Helical Protein.

Proteins are dynamic entities and populate ensembles of conformations. Transitions between states within a conformational ensemble occur over a broad spectrum of amplitude and time scales, and are often related to biological function. Whereas solid-state NMR (SSNMR) spectroscopy has recently been used to characterize conformational ensembles of proteins in the microcrystalline states, its applications to membrane proteins remain limited.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca.

Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR.

The ability to detect and characterize molecular motions represents one of the unique strengths of nuclear magnetic resonance (NMR) spectroscopy. In this study, we report solid-state NMR site-specific measurements of the dipolar order parameters and (15)N rotating frame spin-lattice (R1ρ) relaxation rates in a seven transmembrane helical protein Anabaena Sensory Rhodopsin reconstituted in lipids. The magnitudes of the observed order parameters indicate that both the well-defined transmembrane regions and the less structured intramembrane loops undergo restricted submicrosecond time scale motions.