Backbone structure of a small helical integral membrane protein: A unique structural characterization

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA.
Protein Science (Impact Factor: 2.85). 01/2008; 18(1):134-46. DOI: 10.1002/pro.24
Source: PubMed

ABSTRACT The structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three-dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C-terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 A rmsd backbone structure with a transmembrane domain defined at 0.40 A. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long-range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins.

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Available from: Richard C Page, Aug 16, 2015
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    • "); FXYD1 (Teriete et al., 2007); KCNE1 (Kang et al., 2008); DsbB (Zhou et al., 2008); DAGK, diacylglycerol kinase (Van Horn et al., 2009); Rv1761c (Page et al., 2009); ArcB, QseC, KdpD (Maslennikov et al., 2010); Presenilin-1 C-terminal fragment (Sobhanifar et al., 2010); UCP2, uncoupling protein 2 (Berardi et al., 2011); Proteorhodopsin (Reckel et al., 2011). b Number of conformational restraints used in the structure calculation, as reported in the structural statistics table of the corresponding publication. "
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    ABSTRACT: Nuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data.
    Structure 05/2012; 20(6):1019-27. DOI:10.1016/j.str.2012.03.010 · 6.79 Impact Factor
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    • "Although SDS is a harsh detergent, well-resolved spectra of membrane proteins have been recorded (Howell et al., 2005; Franzin et al., 2007; Teriete et al., 2007). In contrast, DPC and DHPC have a headgroup that closely mimics that of phosphatidylcholine, the most abundant headgroup in natural membranes, successfully used in KcsA (Yu et al., 2005), human phospholamban (Oxenoid & Chou, 2005), diacylglycerol kinase (Van Horn et al., 2009), Rv1761c from Mycobacterium tuberculosis (Page et al., 2009), influenza A M2 (Schnell & Chou, 2008) or HIV Vpu (Park et al., 2003). "
    Human Respiratory Syncytial Virus Infection, 11/2011; , ISBN: 978-953-307-718-5
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    ABSTRACT: The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics of the membrane-spanning peptide. The results make up an important basis for understanding the functional role of proline in several families of membrane proteins.
    Biochemistry 11/2009; 48(50):11883-91. DOI:10.1021/bi9016395 · 3.01 Impact Factor
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