No abstract

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 co-solvent 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, but leads only to correspondingly subtle changes in the spectra. Here we demonstrate that several formulations of GB1 microcrystals yield very high-quality SSNMR spectra, although only a subset of conditions enable growth of single crystals. We have characterized these polymorphs by X-ray powder diffraction and assigned the SSNMR spectra. Assignments of the 13 C and 15 N SSNMR chemical shifts confirm that the backbone structure is conserved, indicative of a common protein fold, but sidechain chemical shifts are changed on the surface of the protein, in a manner dependent upon crystal packing and electrostatic interactions with salt in the mother liquor. Our results demonstrate the ability of SSNMR to reveal minor structural differences among crystal polymorphs. This ability has potential practical utility for studying formulation chemistry of industrial and therapeutic proteins, as well as for deriving fundamental insights into the phenomenon of single crystal growth.

Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21(DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with 13C- and 15N-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins.

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, Escherichia coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled His4CYP98A3 is expressed at yields of 2-4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated His4CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.

Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pK 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-13 C, 15 N-labeled protein were monitored over several samples varying in pH; pK a values were determined from these shifts for E27, D36, and E42, and the bounds for the pK a of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pK a values give insights into the crystal packing of the protein. KeywordsChemical shift perturbation; GB1; Magic-angle spinning; pK a determination; Solid-state NMR Charged residues of a protein play an important role in catalytic reactions, protein stability and substrate binding. [1][2][3] Many enzymatic reactions are controlled by the reversible ionization of charged residues within the active site. This ionization is determined by the intrinsic pK a , which for residues involved in enzymatic mechanisms can deviate greatly from canonical values. 1,[4][5][6] Moreover, pK a values are related to the pH range over which proteins are stable, consistent with the observation of pK a differences among the unfolded and folded proteins, 2,7 with aberrant pK a values often observed in the context of hydrogen bonding, through coupled protonation and deprotonation events. 8 Knowledge of pK a values is useful for purposes of optimizing the absorption and stability of drugs, as well as for identifying substrate interactions in a binding pocket. The central role of ionization events in enzymology has driven the biophysical chemical community to develop methods to accurately determine pK a values across a variety of platforms. 13 Additionally, isotropic chemical shifts were utilized to study rhodopsin and bathorhodopsin and the chromophore retinylidene to gain insights into electrostatic interactions and protonation states in ligation. 14-18 However, these previous SSNMR studies were completed for a small selectively labeled cofactor in the protein systems using one-and two-dimensional experiments, and as such would require many samples in order to assess protonation states of several sites in the protein. Due to the complexity of these issues, no full titration curve to obtain exact pK a values, as routinely done now in solution NMR, has yet been completed utilizing highresolution SSNMR. In this work, we demonstrate that SSNMR can determine both specific pK a values and set bounds for multiple acidic residues in a uniformly labeled protein using multidimensional homo and heteronuclear experiments. The pK a values determined in this work give insight of the protein stability and packing of β1 immunoglobulin binding domain of protein G (GB1) in a nanocrystal environmen...