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Papers by Heather Frericks Schmidt

Research paper thumbnail of Structural Analysis of Membrane-Bound P450s by Magic-Angle Spinning NMR

Research paper thumbnail of A rapid and robust method for selective isotope labeling of proteins

Methods

Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studi... more 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 13 C-and 15 N-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.

Research paper thumbnail of Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR

Proceedings of The National Academy of Sciences, 2008

Magic-angle spinning (MAS) solid-state NMR (SSNMR) techniques have emerged in recent years for so... more Magic-angle spinning (MAS) solid-state NMR (SSNMR) techniques have emerged in recent years for solving complete structures of uniformly labeled proteins lacking macroscopic order. Strategies used thus far have relied primarily on semiquantitative distance restraints, analogous to the nuclear Overhauser effect (NOE) routinely used in solution NMR. Here, we present a complementary approach for using relative orientations of molecular fragments, determined from dipolar line shapes. Whereas SSNMR distance restraints typically have an uncertainty of ≈1 Å, the tensor-based experiments report on relative vector (pseudobond) angles with precision of a few degrees. By using 3D techniques of this type, vector angle (VEAN) restraints were determined for the majority of the 56-residue B1 immunoglobulin binding domain of protein G [protein GB1 (a total of 47 HN-HN, 49 HN-HC, and 12 HA-HB restraints)]. By using distance restraints alone in the structure calculations, the overall backbone root-mean-square deviation (bbRMSD) was 1.01 ± 0.13 Å (1.52 ± 0.12 Å for all heavy atoms), which improved to 0.49 ± 0.05 Å (1.19 ± 0.07 Å) on the addition of empirical chemical shift [torsion angle likelihood obtained from shift and sequence similarity (TALOS)] restraints. VEAN restraints further improved the ensemble to 0.31 ± 0.06 Å bbRMSD (1.06 ± 0.07 Å); relative to the structure with distances alone, most of the improvement remained (bbRMSD 0.64 ± 0.09 Å; 1.29 ± 0.07 Å) when TALOS restraints were removed before refinement. These results represent significant progress toward atomic-resolution protein structure determination by SSNMR, capabilities that can be applied to a large range of membrane proteins and fibrils, which are often not amenable to solution NMR or x-ray crystallography.

Research paper thumbnail of NMR Determination of Protein p K a Values in the Solid State

Journal of Physical Chemistry Letters, 2010

Charged residues play an important role in defining key mechanistic features in many biomolecules... more 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.

Research paper thumbnail of High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

Biochimica Et Biophysica Acta-biomembranes, 2007

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzy... more 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, E. 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 (SAD) 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 13 C-and 15 N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of coexpressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13 C, 15 N-labeled CYP98A3 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 CYP98A3 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.

Research paper thumbnail of Crystal Polymorphism of Protein GB1 Examined by Solid-State NMR Spectroscopy and X-ray Diffraction

Journal of Physical Chemistry B, 2007

The study of micro-or nanocrystalline proteins by magic-angle spinning (MAS) solid-state NMR (SSN... more 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.

Research paper thumbnail of Structural Analysis of Membrane-Bound P450s by Magic-Angle Spinning NMR

Research paper thumbnail of A rapid and robust method for selective isotope labeling of proteins

Methods

Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studi... more 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 13 C-and 15 N-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.

Research paper thumbnail of Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR

Proceedings of The National Academy of Sciences, 2008

Magic-angle spinning (MAS) solid-state NMR (SSNMR) techniques have emerged in recent years for so... more Magic-angle spinning (MAS) solid-state NMR (SSNMR) techniques have emerged in recent years for solving complete structures of uniformly labeled proteins lacking macroscopic order. Strategies used thus far have relied primarily on semiquantitative distance restraints, analogous to the nuclear Overhauser effect (NOE) routinely used in solution NMR. Here, we present a complementary approach for using relative orientations of molecular fragments, determined from dipolar line shapes. Whereas SSNMR distance restraints typically have an uncertainty of ≈1 Å, the tensor-based experiments report on relative vector (pseudobond) angles with precision of a few degrees. By using 3D techniques of this type, vector angle (VEAN) restraints were determined for the majority of the 56-residue B1 immunoglobulin binding domain of protein G [protein GB1 (a total of 47 HN-HN, 49 HN-HC, and 12 HA-HB restraints)]. By using distance restraints alone in the structure calculations, the overall backbone root-mean-square deviation (bbRMSD) was 1.01 ± 0.13 Å (1.52 ± 0.12 Å for all heavy atoms), which improved to 0.49 ± 0.05 Å (1.19 ± 0.07 Å) on the addition of empirical chemical shift [torsion angle likelihood obtained from shift and sequence similarity (TALOS)] restraints. VEAN restraints further improved the ensemble to 0.31 ± 0.06 Å bbRMSD (1.06 ± 0.07 Å); relative to the structure with distances alone, most of the improvement remained (bbRMSD 0.64 ± 0.09 Å; 1.29 ± 0.07 Å) when TALOS restraints were removed before refinement. These results represent significant progress toward atomic-resolution protein structure determination by SSNMR, capabilities that can be applied to a large range of membrane proteins and fibrils, which are often not amenable to solution NMR or x-ray crystallography.

Research paper thumbnail of NMR Determination of Protein p K a Values in the Solid State

Journal of Physical Chemistry Letters, 2010

Charged residues play an important role in defining key mechanistic features in many biomolecules... more 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.

Research paper thumbnail of High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

Biochimica Et Biophysica Acta-biomembranes, 2007

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzy... more 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, E. 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 (SAD) 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 13 C-and 15 N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of coexpressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13 C, 15 N-labeled CYP98A3 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 CYP98A3 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.

Research paper thumbnail of Crystal Polymorphism of Protein GB1 Examined by Solid-State NMR Spectroscopy and X-ray Diffraction

Journal of Physical Chemistry B, 2007

The study of micro-or nanocrystalline proteins by magic-angle spinning (MAS) solid-state NMR (SSN... more 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.