Positioning Membrane Proteins by Novel Protein Engineering and Biophysical Approaches

Abstract

Membrane proteins are unique, in that they can function properly only when they are bound to cellular membranes in a distinct manner. Therefore, positioning of membrane proteins with respect to the membrane is required in addition to the three-dimensional structures in order to understand their detailed molecular mechanisms. Atomic-resolution structures of membrane proteins that have been determined to date provide the atom coordinates in arbitrary coordinate systems with no relation to the membrane and therefore provide little or no information on how the protein would interact with the membrane. This is especially true for peripheral membrane proteins, because they, unlike integral proteins, are devoid of well-defined hydrophobic transmembrane domains. Here, we present a novel technique for determination of the configuration of a protein-membrane complex that involves protein ligation, segmental isotope labeling, polarized infrared spectroscopy, membrane depth-dependent fluorescence quenching, and analytical geometry algorithms. We have applied this approach to determine the structure of a membrane-bound phospholipase A2. Our results provide an unprecedented structure of a membrane-bound protein in which the z-coordinate of each atom is the distance from the membrane center and therefore allows precise location of each amino acid relative to the membrane. Given the functional significance of the orientation and location of membrane-bound proteins with respect to the membrane, we propose to specify this structural feature as the "quinary" structure of membrane proteins.

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

“…A commonly accepted model emerging from those studies is that 1) the enzyme associates peripherally with the macrosubstrate surface, 2) the phospholipid substrate moves upward and engages with the active site cavity of the enzyme, and 3) hydrolysis of the sn2 ester bond takes place, and the products are released from the enzyme (8,18,19,(45)(46)(47)(48)(49)(50). Based on this model, the key factors determining the substrate specificity can be considered to be 1) accommodation of phospholipid acyl chain(s) in the active site cavity of the enzyme and 2) propensity of the phospholipid substrate to efflux from the bilayer.…”

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

“…Production of Recombinant and Semisynthetic Proteins-The semisynthetic, segmentally 13 C-labeled hIBPLA 2 was produced as described previously (18). Briefly, the plasmid for the PLA 2 fragment ⌬N10 that lacks the first 10 residues was constructed by using a human pancreatic cDNA library, and the uniformly 13 C-labeled ⌬N10 was expressed in Escherichia coli BL21(DE3) in an M9 minimal medium using 13 C 6 -Dglucose as a single metabolic source of carbon.…”

“…Briefly, the plasmid for the PLA 2 fragment ⌬N10 that lacks the first 10 residues was constructed by using a human pancreatic cDNA library, and the uniformly 13 C-labeled ⌬N10 was expressed in Escherichia coli BL21(DE3) in an M9 minimal medium using 13 C 6 -Dglucose as a single metabolic source of carbon. The ⌬N10 fragment was purified by ion exchange and size exclusion columns, as described (18). The C-terminally thioesterified N-terminal decapeptide N10hIB was ligated with the N-terminal cysteine of the ⌬N10 fragment, followed by refolding of the ligated PLA 2 by dialysis against 25 mM Tris-HCl, 5 mM CaCl 2 , 5 mM L-cysteine, 0.9 M guanidinium HCl (pH 8.0), and purified by using an ion exchange Mono Q 5/50 column.…”

“…The modeled structure had three ␣-helices, which we call helix 1 (residues 1-10), 2 (residues 43-57), and 3 (91-108). The C-␣ atom coordinates were used to determine the orientations of all three ␣-helices of the protein relative to the "protein" coordinate system ⌺ p with axes x*, y*, z*, in which the coordinates are given, and the helical orientations were further used to determine all interhelical angles, using the analytical geometry algorithms described earlier (18). This indicated that the angle between the helices 2 and 3 was 6.2°, which allowed us to consider these two helices approximately parallel to each other and to use a common order parameter to describe their orientation with respect to the membrane.…”

“…Spectral separation of the ␣-helical signals generated by the unlabeled N-terminal helix and the 13 C-labeled helices 2 and 3 allowed determination of two order parameters, one for the N-terminal helix and one for the helices 2 and 3. Knowledge of the orientations of the two helices in the protein coordinate system ⌺ p and with respect to the membrane normal then allowed determination of the angles between the membrane normal and the three axes of the system ⌺ p , using an algorithm described elsewhere (18). This provides the angular orientation of the protein molecule relative to the membrane.…”