In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 108, No. 37 ( 2011-09-13)
Abstract:
With the addition of more measurements from other techniques, we believe that it will be possible to assess the exact orientation of proteins that interact with biological membranes, and how the binding of additional components affects the orientation, and hence the function, of these complexes. Such studies would improve our understanding of the molecular basis of a variety of cellular processes, particularly those involving the recruitment of signaling molecules to the cell surface. For example, formation of the GRK2-Gβγ complex is thought to play an important role in the progression of heart failure and cardiac hypertrophy. The results obtained in this study represent another step towards a complete understanding of the molecular mechanisms underlying signal transduction at biological interfaces. The tools and methods developed in this research are widely applicable to the analysis of other membrane protein complexes. SFG signals were collected from the proteins associated with the lipid bilayer ( Fig. P1 ) in the so-called Amide I spectral region, which can provide a guide to the overall orientation of a protein backbone. Spectra of the GRK2-Gβγ complex were markedly different from those of the Gβγ subunit alone, and it was shown that the complex could be formed in situ. To assess the orientation of the large GRK2-Gβγ protein complex, the single experimentally observed quantity ( , the ratio of signals from two laser polarizations) was not sufficient. An additional measurement was obtained, based on the observation that the polarized SFG signal intensity from the Gβγ subunit alone was greater than the signal from the entire GRK2-Gβγ complex, which is only possible for certain orientations. It was subsequently determined that the Gβγ subunit reorients to accommodate the binding of GRK2, leading to a membrane orientation consistent with that predicted for a recent and more complete atomic structure of GPCR kinase 6, a GRK2 homolog. Fig. P1. The orientation of the GRK2- Gβ 1 γ 2 complex (inset) can be studied using SFG vibrational spectroscopy. Spectra were collected from samples of protein in contact with a 9∶1 POPC/POPG lipid bilayer. Based on analysis of fitted spectral intensity ratios in spectra collected using different laser polarizations, it was found that the orientation of GRK2- Gβ 1 γ 2 did not match that predicted from the crystal structure alone. A possible membrane orientation (shown) is consistent with the presence of a GPCR docking site homology modeled from the structure of GRK6. The protein, lipid bilayer, SFG beams, the prism, and the sample container are not drawn to scale. This program was applied to study the orientation of a multisubunit protein-protein complex, specifically the G protein-coupled receptor kinase 2 (GRK2)- Gβ 1 γ 2 complex. GRK2 is a kinase enzyme responsible for regulation of cell surface G protein-coupled receptors (GPCRs) signaling. Because of its apparent ability to simultaneously interact with activated G proteins, GPCRs, and the membrane, GRK2 may be involved in the assembly and organization of signaling complexes at GPCRs. Although the atomic structure of GRK2 in complex with both Gα q and Gβγ is known ( 4 ), its orientation while engaged at the cell membrane is less clear because many of the known membrane-binding determinants in this structure are disordered. Previously, we studied the orientation of the smaller Gβγ subunit alone at the bilayer ( 5 ), but it is possible that this subunit changes orientation to accommodate the binding of GRK2. In this work, we sought to determine the orientation of GRK2 and Gβγ relative to the bilayer and to each other, providing an in situ glimpse of molecular orientation and complex formation. To date, however, studies on the orientation of large biomolecules have been hampered by the difficulty of interpreting their complicated spectra. Although a protein may contain multiple segments of spiral-shaped motifs called alpha-helices, these regions collectively contribute to a single vibrational peak center in the SFG spectrum. Thus efforts to determine the overall molecular orientation depend on the combined molecular response for all helical segments. We have developed a computer program that greatly simplifies the analysis of SFG data for any arbitrary protein structure, and that determines the most likely orientations of the protein at a membrane surface. It has been estimated that as many as half of all proteins in a typical cell interact with the cell membrane, yet most tools used to study biomolecules require removing those molecules from their native environment. This requirement is partly due to a lack of surface sensitive techniques capable of studying biomolecules in situ in an aqueous lipid environment. A technique called Sum Frequency Generation (SFG) vibrational spectroscopy ( 1 ) was recently used to reveal the spectra of small peptides and proteins at solid/liquid interfaces or in lipid bilayers ( 2 , 3 ), thereby providing information about molecular orientation, secondary structure, and chemical functional groups.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1108236108
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2011
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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