One of the main applications of solid-state NMR is to study the structure and dynamics of biopolymers, such as membrane proteins, under physiological conditions where the polypeptides undergo global motions as they do in biological membranes. The effects of NMR radiofrequency irradiations on nuclear spins are strongly influenced by these motions. For example, we previously showed that the MSHOT-Pi4 pulse sequence yields spectra with resonance line widths about half of those observed using the conventional pulse sequence when applied to membrane proteins undergoing rapid uniaxial rotational diffusion in phospholipid bilayers. In contrast, the line widths were not changed in microcrystalline samples where the molecules did not undergo global motions. Here, we demonstrate experimentally and describe analytically how some Hamiltonian terms are susceptible to sample motions, and it is their removal through the critical π/2 Z-rotational symmetry that confers the "motion adapted" property to the MSHOT-Pi4 pulse sequence. This leads to the design of separated local field pulse sequence "Motion-adapted SAMPI4" and is generalized to an approach for the design of decoupling sequences whose performance is superior in the presence of molecular motions. It works by cancelling the spin interaction by explicitly averaging the reduced Wigner matrix to zero, rather than utilizing the 2π nutation to average spin interactions. This approach is applicable to both stationary and magic angle spinning solid-state NMR experiments.

Oriented sample solid-state NMR spectroscopy can be used to determine the three-dimensional structures of membrane proteins in magnetically or mechanically aligned lipid bilayers. The bottleneck for applying this technique to larger and more challenging proteins is making resonance assignments, which is conventionally accomplished through the preparation of multiple selectively isotopically labeled samples and performing an analysis of residues in regular secondary structure based on Polarity Index Slant Angle (PISA) Wheels and Dipolar Waves. Here we report the complete resonance assignment of the full-length mercury transporter, MerF, an 81-residue protein, which is challenging because of overlapping PISA Wheel patterns from its two trans-membrane helices, by using a combination of solid-state NMR techniques that improve the spectral resolution and provide correlations between residues and resonances. These techniques include experiments that take advantage of the improved resolution of the MSHOT4-Pi4/Pi pulse sequence; the transfer of resonance assignments through frequency alignment of heteronuclear dipolar couplings, or through dipolar coupling correlated isotropic chemical shift analysis; (15)N/(15)N dilute spin exchange experiments; and the use of the proton-evolved local field experiment with isotropic shift analysis to assign the irregular terminal and loop regions of the protein, which is the major "blind spot" of the PISA Wheel/Dipolar Wave method.

In the stationary, aligned samples used in oriented sample (OS) solid-state NMR, (1)H-(1)H homonuclear dipolar couplings are not attenuated as they are in magic angle spinning solid-state NMR; consequently, they are available for participation in dipolar coupling-based spin-exchange processes. Here we describe analytically the pathways of (15)N-(15)N spin-exchange mediated by (1)H-(1)H homonuclear dipolar couplings. The mixed-order proton-relay mechanism can be differentiated from the third spin assisted recoupling mechanism by setting the (1)H to an off-resonance frequency so that it is at the "magic angle" during the spin-exchange interval in the experiment, since the "magic angle" irradiation nearly quenches the former but only slightly attenuates the latter. Experimental spectra from a single crystal of N-acetyl leucine confirm that this proton-relay mechanism plays the dominant role in (15)N-(15)N dilute-spin-exchange in OS solid-state NMR in crystalline samples. Remarkably, the "forbidden" spin-exchange condition under "magic angle" irradiation results in (15)N-(15)N cross-peaks intensities that are comparable to those observed with on-resonance irradiation in applications to proteins. The mechanism of the proton relay in dilute-spin-exchange is crucial for the design of polarization transfer experiments.

Near-infrared surface plasmon resonance imaging (SPRI) microscopy is used to detect and characterize the adsorption of single polymeric and protein nanoparticles (PPNPs) onto chemically modified gold thin films in real time. The single-nanoparticle SPRI responses, Δ%R_NP, from several hundred adsorbed nanoparticles are collected in a single SPRI adsorption measurement. Analysis of Δ%R_NP frequency distribution histograms is used to provide information on the size, material content, and interparticle interactions of the PPNPs. Examples include the measurement of log-normal Δ%R_NP distributions for mixtures of polystyrene nanoparticles, the quantitation of bioaffinity uptake into and aggregation of porous NIPAm-based (N-isopropylacrylamide) hydrogel nanoparticles specifically engineered to bind peptides and proteins, and the characterization of the negative single-nanoparticle SPRI response and log-normal Δ%R_NP distributions obtained for three different types of genetically encoded gas-filled protein nanostructures derived from bacteria.

MerF is a mercury transport membrane protein from the bacterial mercury detoxification system. By performing a solid-state INEPT experiment and measuring chemical shift anisotropy frequencies in aligned samples, we are able to improve on the accuracy and precision of the initial structure that we presented. MerF has four N-terminal and eleven C-terminal residues that are mobile and unstructured in phospholipid bilayers. The structure presented here has average pairwise RMSDs of 1.78 Å for heavy atoms and 0.92 Å for backbone atoms.