66 1682009)61-7元 the ent meters sohcdntheequengydomaiandtheeioteheo family of peripheral membrane proteins that tran sduce extracelly ar signals (e.g d by . GPCRS to intr In:-Ay+ (6) tem s(e ptor d ce subunits,with a=m+远 com prised GB.and G Gy forming the form.Upon GPCR activation AsEqs.()-(5)indicate.nly wo theri tion of nto Ga-GTP and et a.1999 the luced.If all elical structures o s The ient nd co ubsequen 业.the DP (200 for most cases.it ma her et al.. y no and al.2007ai tigated hoy interface have the same orientation. one unit ents on a substrate-sup e u structures omplex case ed popo bilayer. ple ed minode pe Che tion.These two distributions we therefore enh ancement of nal that was dominated by Cher 8-distrbution as the he s th A fraction(N)o mol es ng more llel to itti the ar to the b fully deduced.The ATR-FTIRexp to the urface keep its ulations in th ut th of melitti and as shown ng and B deduced the orientation ange melittin in a si wit to the me ATR ETIR th umptio s reg ling the SFG can be used to deduce the m protein orientatio Thededcaomentaiomdsnbuic components of usin g the maximum en the d 3.3.3.SFG studies on 3 helical peptides in membrane bilaver ysis ments can form voltage-gated ion cha as beer tides and utimate lead to and In addit nto the re ds,th ctivit
where baca and bccc are the molecular hyperpolarizability elements. The hyperpolarizability elements of an a-helix can be obtained from the product of the components of the Raman polarizability and IR transition dipole moment. Chen et al. (Chen et al., 2007c) deduced the relations among different hyperpolarizability tensor elements to be r = baac/bccc0.54 and baca0.32 bccc (Lee and Krimm, 1998a,b; Marsh et al., 2000; Rintoul et al., 2000). Ns is the number density of ideal a-helix units composed of 18 amino acid residues. Due to the limited resolution of many SFG spectrometers (5 cm1 or more), the A mode and E1 mode cannot be readily resolved in the frequency domain, and therefore, the total susceptibility is often assumed to be the sum of the susceptibilities from these two modes (Chen et al., 2007c): vyyz ¼ vA;yyz þ vE1;yyz ð6Þ vzzz ¼ vA;zzz þ vE1;zzz ð7Þ As Eqs. (2)–(5) indicate, only two measurables related to the orientation angle are independent: <cosh> and <cos3 h>. Using different polarization combinations of the input and output laser beams, <cosh> and <cos3 h> can be deduced. If all a-helical structures on the surface/interface adopt the same orientation, <cosh> and <cos3 h> can be replaced by cosh and cos3 h. Subsequently, the relationship between an SFG measurable and the orientation angle of the a-helix can be depicted. However, for most cases, it may not be correct to assume that all the a-helical structures on a surface/ interface have the same orientation. For example, one protein may have two a-helical segments pointing two different directions. The following section discusses the orientation analysis of a-helical structures in some of these complex cases. 3.3.2.2. Example: a-helical melittin in membrane. Recently, Chen et al. used melittin as a model peptide to study the orientation of a-helical peptides in substrate-supported DPPG bilayers (Chen et al., 2007c). Chen et al. found that the SFG measurements were not compatible to those of a d-distribution or a Gaussian distribution. These two distributions were therefore not adequate to describe the melittin orientation distribution inside a DPPG bilayer and the orientation distribution had to be more complex. Chen et al. assumed two d-distributions as the orientation function, meaning that melittin was assumed to adopt two distinct orientations in the lipid bilayer. A fraction (N) of melittin molecules may orient with an angle of h1, and a fraction (1-N) of melittin molecules can orient with another angle of h2. By combined ATR-FTIR and SFG studies, all of these parameters, h1, h2 and N, were successfully deduced. The obtained results from SFG and ATR-FTIR experiments indicated that melittin helices existed in two main populations in the lipid bilayer. About three–fourths of melittin molecules oriented parallel to the bilayer surface with a slight tilt, while the rest oriented more or less parallel to the surface normal, as shown in Fig. 2A and B. In addition, Chen et al. also introduced the maximum entropy function to deduce the orientation distribution of melittin in a single lipid bilayer based on the ATR-FTIR and SFG measurements. Such a treatment does not have any assumptions regarding the orientation distribution function, e.g., assuming two d-distributions. The deduced orientation distribution using the maximum entropy function was very similar to that obtained from the two d-distributions, as shown in Fig. 2C. This research demonstrated the power of combining ATR-FTIR measurements, SFG data and the maximum entropy function analysis for deducing complicated orientations of membrane-bound peptides. These kinds of orientation determination results can be correlated to different modes of action of peptides’ interactions with bilayers, and ultimately lead to an understanding of the mechanism of antimicrobial activity, for example. Such an analysis can also be applied to study interfacial proteins with two (or even three) a-helical segments that adopt different orientations. A similar method has also been used to study the orientation distribution of two a-helical coiled-coils of fibrinogen at the polystyrene/fibrinogen solution (phosphate buffered solution with a total ionic strength of 0.14 M and a pH value of 7.4) interface (Wang et al., 2008). 3.3.2.3. Example: a-helical structure in G protein in lipid bilayer. Heterotrimeric guanine nucleotide-binding proteins (G proteins) are a family of peripheral membrane proteins that transduce extracellular signals (e.g., hormones and neurotransmitters), as sensed by G protein-coupled-receptors (GPCRs), to intracellular effector systems (e.g., ion channels and cell transcription machinery) (Cabrera-Vera et al., 2003; Neves et al., 2002). Each G protein is comprised of Ga, Gb, and Gc subunits, with Gb and Gc forming a tightly associated dimer. In the resting state, a G protein exists in the Gabc form. Upon GPCR activation, Gabc releases the GDP originally bound to the Ga subunit and the binding of GTP allows dissociation of Gabc into GaGTP and Gbc (Gaudet et al., 1999; Lodowski et al., 2003). Ga and Gbc can then associate with their own effectors and trigger downstream signaling cascades. The cycle returns to the resting state when Ga hydrolyzes GTP back to GDP (Koch, 2004; Neves et al., 2002; Oldham and Hamm, 2006; Pitcher et al., 1992). Recently, (Chen et al., 2007a) investigated how the Gb1c2 subunit binds to and orients on a substrate-supported lipid bilayer using SFG. Both wild-type Gb1c2 subunits, which contain a geranylgeranyl anchor group, and only the soluble domain of the Gb1c2 subunits were used in this research. SFG spectra were collected from both types of G protein subunits in a hydrated POPG/ POPG bilayer. These two Gb1c2 subunits showed very different SFG spectral properties (Fig. 3C and D). Even at relatively high concentrations (125 lg/mL), soluble Gb1c2 generated weaker signals than geranylgeranylated Gb1c2, with a peak centered at 1630 cm1 , indicative of b-sheet secondary structure. The presence of the geranylgeranyl anchor group resulted in a significant enhancement of SFG amide I signal that was dominated by a peak at around 1650 cm1 , which is characteristic of contributions from an a-helical structure. (Chen et al., 2007a) suggested that without the geranylgeranyl group, Gb1c2 adsorbs onto the surface with the b-propeller domain facing the membrane surface and the helical domains orienting more or less parallel to the surface (Fig. 3B, Chen et al., 2007a). On the other hand, for the wild-type Gb1c2 subunit, the b-propeller more or less orients perpendicular to the bilayer surface and the helical domains are ordered and no longer parallel to the surface. This orientation allows the b-propeller to keep its native semi-centrosymmetry, resulting in very weak b-sheet signal and causing the amide I signal to be dominated by a peak at 1650 cm1 (originating from the ordered helical domains), as shown in Fig. 3A. From the measured SFG ppp and ssp intensity ratio, Chen et al. deduced the orientation angle of the wild-type Gb1c2 to be 35 from a reference orientation in which the bsheets within the b-propeller are parallel to the membrane surface (Tesmer et al., 2005; Wall et al., 1995). This research demonstrates that SFG can be used to deduce the membrane protein orientation in situ by studying the orientation of a-helical components of a protein. 3.3.3. SFG studies on 310 helical peptides in membrane bilayer: alamethicin Alamethicin is a 20-residue hydrophobic antibiotic peptide that can form voltage-gated ion channels in membranes. It has been used frequently as a model for larger channel proteins (Tamm and Tatulian, 1997). In addition to the regular amino acids, the peptide contains eight aminoisobutyric acid units. Its crystal struc- 66 S. Ye et al. / Journal of Structural Biology 168 (2009) 61–77
A Solid substrate 了万万万下奶万万万万万万万万万 Lipid 31题55从 Bilayer Melittin B 01 0.5 0 30 60 90 120 150 angle(degree) C0.07 0.06 0.03 0.0 0 30 60 90 120 150 180 angle(degree) 二品 ic membrane nport ewski,001;Hall et a9 4:Leitgel onductivity on the transmembrane potential(Stella et)
ture contains an a-helical domain and a 310 helical domain (Fox and Richards, 1982). An extensive amount of research has been performed to examine the alamethicin action mechanism on membranes (Cafiso, 1994; Duclohier and Wroblewski, 2001; Hall et al., 1984; Leitgeb et al., 2007; Mathew and Balaram, 1983a,b; Nagaraj and Balaram, 1981; Sansom, 1993a,b; Woolley and Wallace, 1992). It is currently believed that alamethicin interacts with cell membranes through the barrel-stave mode (Duclohier, 2004; Fox and Richards, 1982; Laver, 1994; Mathew and Balaram, 1983a,b; Sansom, 1993a,b) with the resulting conducting pores in the membrane formed by parallel bundles of 3–12 helical alamethicin monomers surrounding a central, water-filled pore. However, further details on the structural origin of some important properties of alamethicin channels in the membrane, such as the strong dependence of their conductivity on the transmembrane potential (Stella et al., 2007), are not known. In addition, contradicting orientations of alamethicin in the membrane in the absence of voltage have been reported. Alamethicin has been suggested to adopt a transmembrane orientation (Bak et al., 2001; Kessel et al., 2000; Marsh et al., 2007a; North et al., 1995), lie on the membrane surface (Banerjee et al., 1985; Ionov et al., 2000; Mottamal and Lazaridis, 2006), or both 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 30 60 90 120 150 180 0 30 60 90 120 150 180 angle (degree) Population 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 angle (degree) Population A B C Fig. 2. (A) Schematic of melittin’s two orientations in the lipid bilayer. (B) Orientation distribution function derived based on a dual d-distribution. (C) Orientation distribution function derived based on the maximum entropy theory. Reproduced with permission from J. Am. Chem. Soc. 2007, 129, 1420–1427. Copyright 2007, American Chemical Society. S. Ye et al. / Journal of Structural Biology 168 (2009) 61–77 67