Kurt W. Zilm

The function of a protein is predicated upon its three-dimensional fold. Representing its complex structure as a series of repeating secondary structural elements is one of the most useful ways by which we study, characterize, and visualize a protein. Consequently, experimental methods that quantify the secondary structure content allow us to connect a protein's structure to its function. Here, we introduce an automated gradient descent-based method we refer to as secondary-structure distribution by NMR that allows for rapid quantification of the protein secondary structure composition of a protein from a single, 1D ¹³C NMR spectrum without chemical shift assignments. The analysis of nearly 900 proteins with known structure and chemical shifts demonstrates the capabilities of our approach. We show that these results rival alternative techniques such as FT-IR and circular dichroism that are commonly used to estimate secondary structure compositions. The resulting method requires only the primary sequence of the protein and its referenced ¹³C NMR spectrum. Each residue is modeled in an ensemble of secondary structures with percentage contributions from random coil, α-helix, and β-sheet secondary structures obtained by minimizing the difference between a simulated and experimental 1D ¹³C NMR spectrum. The capabilities of the method are demonstrated as applied to samples at natural abundance or enriched in ¹³C, acquired by either solution or solid-state NMR, and even on low magnetic field benchtop NMR spectrometers. This approach allows for rapid characterization of protein secondary structure across traditionally challenging to characterize states including liquid-liquid phase-separated, membrane-bound, or aggregated states.

The function of a protein is predicated upon its three dimensional fold. Representing its complex structure as a series of repeating secondary structural elements is one of the most useful ways by which we study, characterize, and visualize a protein. Consequently, experimental methods that quantify the secondary structure content allow us to connect a protein’s structure to its function. Here, we introduce an automated gradient descent-based method we refer to as Secondary Structure Distribution by NMR that allows for rapid quantification of the protein secondary structure composition of a protein from a single, 1D 13C NMR spectrum without chemical shift assignments. The analysis of nearly 900 proteins with known structure and chemical shifts demonstrates the capabilities of our approach. We show that these results rival alternative techniques such as FT-IR and circular dichroism that are commonly used to estimate secondary structure compositions. The resulting method requires only the primary sequence of the protein and its referenced 13C NMR spectrum. Each residue is modeled in an ensemble of secondary structures with percentage contributions from random coil, α-helix, and β-sheet secondary structures obtained by minimizing the difference between a simulated and experimental 1D 13C NMR spectrum. The capabilities of the method are demonstrated as applied to samples at natural abundance or enriched in 13C, acquired by either solution or solid-state NMR, and even on low magnetic field benchtop NMR spectrometers. This approach allows for rapid characterization of protein secondary structure across traditionally challenging to characterize states including liquid-liquid phase-separated, membrane-bound, or aggregated states.

The difficulties in quantitatively modeling the temperature dependence of spin–lattice relaxation in a model isotope-enriched peptide are explored as a prelude to obtaining dynamics parameters for motions in proteins from such measurements. The degree to which this can be handled by adding spin diffusion to a bath in standard rate matrix relaxation theory is studied using a small tri-peptide model system, glycyl–alanyl-leucine (GAL). We observe in this molecule that the relaxation of backbone carbons CO and Cα is not dominated by local fluctuations of the ¹³C–¹H dipolar couplings, but rather by ¹³C–¹³C spin diffusion to nearby methyl relaxation sinks. A treatment of the methyl relaxation itself, which ignores ¹³C–¹³C spin diffusion effects back to the otherwise slowly relaxing bath, provides poor agreement between theory and experimental data obtained for the temperature dependence of the methyl relaxation rates. Closed form approximate spectral densities and relaxation rates for a methyl group during magic angle spinning are obtained to compute the needed transition rates. These average computed rates, in conjunction with an extended form of the Solomon equations, are found to adequately model the temperature dependence of the methyl relaxation rates when spin diffusion is included. The barrier to rotation for the alanine methyl in GAL is determined to be 3.5 kcal mol⁻¹.

Infectious mammalian prions can be formed de novo from purified recombinant prion protein (PrP) substrate through a pathway that requires the sequential addition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and RNA cofactor molecules. Recent studies show that the initial interaction between PrP and POPG causes widespread and persistent conformational changes to form an insoluble intermediate species, termed PrP^Int1. Here, we characterize the mechanism and functional consequences of the interaction between POPG and PrP. Negative-stain electron microscopy of PrP^Int1 revealed the presence of amorphous aggregates. Pull-down and photoaffinity label experiments indicate that POPG induces the formation of a PrP^C polybasic-domain-binding neoepitope within PrP^Int1. The ongoing presence of POPG is not required to maintain PrP^Int1 structure, as indicated by the absence of stoichiometric levels of POPG in solid-state NMR measurements of PrP^Int1. Together, these results show that a transient interaction with POPG cofactor unmasks a PrP^C binding site, leading to PrP^Int1 aggregation.

A surprisingly strong spin rate dependence of ¹⁵N and ¹³C NMR T₁ times in magic angle spinning experiments on solid peptides is demonstrated. Using a variety of isotopomers, the phenomenon is shown to be the result of chemical shift anisotropy-mediated spin diffusion. This effect has the potential to be used to detect long-range distance constraints in macromolecular systems.

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at ∼20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only ∼0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.

The relative merits of different isotopic enrichment strategies that might be used in solid state NMR protein structure determinations are explored. The basis for comparison of these merits is the...