Hydropathy analysis

FIGURE 10.5 A model for the arrangement of the glucose transport protein in the erythrocyte membrane. Hydropathy analysis is consistent with 12 transmembrane helical segments. 

M FIGURE 10.14 The arrangement of Ca -ATPase in the sarcoplasmic reticulum membrane. Ten transmembrane segments are postulated on the basis of hydropathy analysis. 

Information about the putative folding of the H,K-ATPase catalytic subunit through the membrane has been obtained by the combined use of hydropathy analysis according to the criteria of Kyte and Doolittle [51], identification of sites sensitive to chemical modification [46,48,50,52-55], and localization of epitopes of monoclonal antibodies [56]. The model of the H,K-ATPase catalytic subunit (Fig. 1) which has emerged from these studies shows ten transmembrane segments and contains cytosolic N- and C-termini [53]. This secondary structure of the catalytic subunit is probably a common feature of the catalytic subunits of P-type ATPases, since evidence supporting a ten a-helical model with cytosolic N- and C-termini has also been published recently for both Ca-ATPase of the sarcoplasmic reticulum and Na,K-ATPase [57-59]. 

Hydropathy analysis predicted that there are four major transmembrane domains (M1-M4) prior to the phosphorylation site at Asp . The existence of these four transmembrane segments in the N-terminal half of the catalytic subunit is generally accepted for all P-type ATPases. The four transmembrane sequences are followed by a large cytosolic loop that contains the phosphorylation site Asp, the pyridoxal... 

With this information in hand, we may now consider how the -ATPase polypeptide chain might fold into its functional three-dimensional structure. First, regarding the actual number of membrane-spanning stretches, the available experimental data indicate only that each of the three membrane-embedded peptides must have an even number of and a minimum of two such stretches. However, hydropathy analysis by the method of Mohana Rao and Argos [48] suggests that the second mem-... 

From a procedural standpoint, the start and stop residues of each of the seven helices was first identified by hydropathy analysis by using Kyte-Doolittle19 hydropathy indices. Each of the seven helices was then built individually, essential hydrogens were added, and the peptides were minimized with the Kollman United Atoms force field within the Sybyl software suite (Tripos Associates, Inc.). Typically, steepest descent was used initially as the minimization method (25-50... 

In addition to the high sequence homology of these carriers, secondary structure models based on hydropathy analysis (Amara and Kuhar, 1993) are virtually superimposable. All have been assigned 12 transmembrane domains and each transporter is considered to have a large extracellular glycosylated loop between transmembrane domains three and four. 

Figure 5 shows the structure of the dimer of cytochrome b in the bc complex. Both the N-terminus and the C-terminus of the subunit are located in the matrix side. The eight transmembrane helices are approximately at the same places as predicted from hydropathy analysis and mutagenesis studies (Link et ah, 1994 Crofts et al., 1992). The helices are connected by four long linkers (AB, CD, DE, and EF) and three short ones (BC, FG, and GH).The AB and EF linker eaeh contains one a helix, labeled as ab and ef in Figure 5. The CD linker has two short helices cdl and cd2, which form a hairpin structure. The DE linker has no regular secondary structure. In the ehieken complex, helix F was bent by over 45 in the middle around residue 296. A short amphipathie helix a, preceding all the transmembrane heliees, runs parallel to the membrane.

Ste24p is predicted to have seven transmembrane spans by hydropathy analysis. Experimental evidence supports the view that the N-terminus of Ste24p is luminal and its C-terminus is cytsolic, consistent with the predicted odd number of spans [54]. The C-terminus of Ste24p contains a dilysine ER retrieval motif (KKXX), and the protease is dually localized to the ER and the INM [5,48,58]. Importantly, the Ste24p zinc metalloprotease motif (HEXXH), which lies within a large cytosolic loop between the two C-terminal membrane spans, is appropriately cytosolically positioned for processing of a membrane-bound prenylated substrate such as a-factor. 

Secondary transporters are ancient molecular machines, common today in bacteria and archaea as well as in eukaryotes. For example, approximately 160 (of approximately 4000) proteins encoded by the E. coli genome appear to be secondary transporters. Sequence comparison and hydropathy analysis suggest that members of the largest family have 12 transmemhrane helices that appear to have arisen by duplication and fusion of a membrane protein with 6 transmemhrane helices. Included in this family is the lactose permease of E. coli. This symporter uses the H+ gradient... 

Fig. 1 Primary amino acid sequence comparison between five members of the human somatostatin receptor family. The amino acid sequences are aligned, using the single-letter amino acid code. Gaps introduced in the sequences to optimize the alignments are represented with dashes (-). The boxes indicate amino acids that are identical in all five receptor subtypes. The highest level of homology occurs within the seven canonical transmembrane domains of the human SST receptors (indicated above the sequences) and these domains have been derived by a combination of hydropathy analysis and comparison with the transmembrane domains of the other G-protein coupled receptors. The high level of homology among the members of the SST receptor family in the seven membrane spanning domains is shared by other G-protein coupled receptors that are coupled to inhibition of adenylyl cyclase.

Oatps/OATPs are key membrane transporters for which crystal structures are not available. According to the hydropathy analysis, all Oatps/OATPs contain 12 transmembrane domains with both the amino and the carboxy terminal parts located intracellulary (Figure 3.2). However, the predicted 12-transmembrane domain model for any Oatp/OATP has not been proven experimentally [8]. 

Structure-Transport Relationship of PepTI. Structural information on PepTl has been limited to its primary sequence and predicted structural membrane topology. Hydropathy analysis of the human, rabbit, and rat PepTl isoforms have predicted the presence of 12 transmembrane domains (TMD) in each isoform (212). This model has been partially proved by other investigators (144,213). 

Figure 8.10. Membrane topology of ASBT. Hydropathy analysis proposes a seven transmembrane topology for ASBT. Studies with antibodies to terminal epitopes have confirmed the inside-out orientation of this membrane protein.

Hydropathy analysis indicates that Algl contains at least 10 transmembrane helices, and experimentally it has been demonstrated to be associated with the inner membrane. These properties suggest that Algl may be... 

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