Membrane protein folding: beyond the two stage model

256

279

The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd <1.5 Å), allowing 90% of the library to be assigned to clusters consisting of at least five members. Surprisingly, three quarters of the helical pairs belong to one of five tightly clustered motifs whose structural features can be understood in terms of simple principles of helix-helix packing. Thus, the universe of common transmembrane helix-pairing motifs is relatively simple. The largest cluster, which comprises 29% of the library members, consists of an antiparallel motif with left-handed packing angles, and it is frequently stabilized by packing of small side chains occurring every seven residues in the sequence. Righthanded parallel and antiparallel structures show a similar tendency to segregate small residues to the helix-helix interface but spaced at four-residue intervals. Position-specific sequence propensities were derived for the most populated motifs. These structural and sequential motifs should be quite useful for the design and structural prediction of membrane proteins.helix-helix packing ͉ protein design ͉ structure prediction H elical transmembrane (TM) proteins are a major class of membrane proteins that are critically involved in functionally rich processes, including bioenergetics, signal transduction, ion transmission, and catalysis. The mechanisms by which TM proteins fold into native structures are beginning to be understood from a confluence of structural and biochemical studies (1-3). The determinants of a membrane protein's fold can be understood by dissecting its structure into pairs of interacting helices, which, together with the connecting loops and extramembrane domains, comprise the overall structure. Along these lines, various workers have examined the geometric characteristics of helix-helix interactions in membrane proteins (4) as well as the features in the amino acid sequences that predispose the helices to adopt these geometries. One outstanding success has been the recognition of the GX 3 G motif, in which Gly (or other small residues) spaced four residues apart mediate a close approach of TM helices (5-7). This motif was first observed in the TM domain of glycophorin A (GpA) (5) and has subsequently been found in both water-soluble (8) and membrane proteins (9). In the classical GpA GX 3 G motif, the helices cross with a right-handed crossing angle of Ϸ40°. In a different TM motif stabilized by ''knobs-into-holes'' packing (10), the helices cross with a smaller left-handed crossing angle.Other surveys of helix-helix packing in membrane proteins have focused on distributions of the interhelical angles, distances, and the composition of the side chains packed at the interface. The helix-helix interfaces tend to be well pa...

Section: Clusters 1 and 4: Antiparallel And Parallel Pairs With Left-supporting

confidence: 88%

Section: Different Motifs Have Different Numbers Of Residues Between Thementioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Helix-packing motifs in membrane proteins

Walters¹,

DeGrado

2006

256

279

“…The interactions with the membranes or membrane-mimicking systems induce a stable ␣-helical conformation. This process is reminiscent of the membrane association mechanisms proposed for small hormone or antimicrobial peptides (48) and of the prefolding states of membrane proteins identified by Engelman and coworkers (49,50).…”

Section: Discussionmentioning

confidence: 74%

Spectroscopic validation of the pentameric structure of phospholamban

Traaseth¹,

Verardi

Torgersen

et al. 2007

126

Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA) affinity for calcium. Although the monomeric form of PLN (6 kDa) is the principal inhibitory species, recent evidence suggests that the PLN pentamer (30 kDa) also is able to bind SERCA. To date, several membrane architectures of the pentamer have been proposed, with different topological orientations for the cytoplasmic domain: (i) extended from the bilayer normal by 50 -60°; (ii) continuous ␣-helix tilted 28°relative to the bilayer normal; (iii) pinwheel geometry, with the cytoplasmic helix perpendicular to the bilayer normal and in contact with the surface of the bilayer; and (iv) bellflower structure, in which the cytoplasmic domain helix makes Ϸ20°angle with respect to the membrane bilayer normal. Using a variety of cell membrane mimicking systems (i.e., lipid vesicles, oriented lipid bilayers, and detergent micelles) and a combination of multidimensional solution/solidstate NMR and EPR spectroscopies, we tested the different structural models. We conclude that the pinwheel topology is the predominant conformation of pentameric PLN, with the cytoplasmic domain interacting with the membrane surface. We propose that the interaction with the bilayer precedes SERCA binding and may mediate the interactions with other proteins such as protein kinase A and protein phosphatase 1.Ca 2ϩ -ATPase ͉ EPR ͉ membrane protein ͉ solid-state NMR ͉ protein dynamics C alcium translocation into the sarcoplasmic reticulum of cardiac myocytes is controlled by the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA). Phospholamban (PLN) regulates the activity of SERCA by shifting the apparent calcium affinity for the enzyme. This activity is relieved by phosphorylation of PLN at Ser-16 and/or Thr-17 and high calcium concentration within the cytosol. Wild-type PLN (wt-PLN) forms stable homopentamers in lipid bilayers and in detergent micelles, where each monomer is composed of a helical cytoplasmic domain (residues 1-16), a semiflexible loop (residues 17-21), and a helical transmembrane domain (residues 22-52) (1, 2). Mutagenesis, molecular biology, and in vivo studies revealed that the PLN pentamer depolymerizes into active monomers that bind and inhibit SERCA (3). Similar conclusions were reached by in vitro fluorescence studies (4). Recently, Young and coworkers (5) have reported a cocrystal formed by SERCA and PLN pentamer, suggesting that the pentameric species also is able to bind SERCA. Furthermore, Jones and coworkers (6) hypothesized that the PLN pentamer may act as a chloride ion channel, which is supported by the bellflower structure recently determined by Oxenoid and Chou (2).There are four principal proposed structural models of pentameric wt-PLN, which differ primarily in the topology of the more dynamic cytoplasmic domain. In each of these models (shown in Fig. 1), residues 32-52 are in a coiled helix approximately parallel to the bilayer normal. The first model (extended helix/sheet ...

“…thermodynamics ͉ drug delivery ͉ imaging ͉ membrane protein T he folding of helical membrane proteins can be conceptualized in terms of distinct steps: (i) peptide insertion and folding to form independently stable helices across a lipid bilayer, (ii) helix association to form a helix bundle intermediate, and (iii) further rearrangements of protein structure and/or binding of prosthetic groups to achieve a functional state (1). Insertion of most membrane proteins is facilitated in vivo by complex molecular machines, such as the translocon, which assist in placing hydrophobic sequences across the bilayer (2).…”

mentioning

confidence: 99%

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Reshetnyak

Andreev

Segala

et al. 2008

Self Cite

164

180

The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric ␣-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I-state II transition) at 37°C is approximately ؊7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II-state III transition) is nearly ؊2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.thermodynamics ͉ drug delivery ͉ imaging ͉ membrane protein T he folding of helical membrane proteins can be conceptualized in terms of distinct steps: (i) peptide insertion and folding to form independently stable helices across a lipid bilayer, (ii) helix association to form a helix bundle intermediate, and (iii) further rearrangements of protein structure and/or binding of prosthetic groups to achieve a functional state (1). Insertion of most membrane proteins is facilitated in vivo by complex molecular machines, such as the translocon, which assist in placing hydrophobic sequences across the bilayer (2). In contrast to more hydrophobic sequences, moderately polar transmembrane domains of Cterminally-anchored proteins can postranslationally translocate themselves into membranes in a translocon-defective yeast strain (3, 4), seeking the free-energy minimum (5). Thus, insertion of a disordered peptide in aqueous solution to form a transbilayer ␣-helix is accompanied by a release of energy, so thermodynamic studies of the interaction of proteins and peptides with a lipid bilayer can provide insights regarding folding. However, such studies are significantly complicated by the poor solubility of membrane peptides, which has made it difficult to separate the contributions of peptide interactions with a lipid bilayer from self-interactions and aggregation.We have been studying a useful system for measurement of the thermodynamics and kinetics of peptide insertion and folding across a lipid bilayer. It is based on the pH low-insertion peptide (pHLIP), which has three major states: (I) soluble in water in an unstructured, monomeric state, (II) bound to the surface of a lipid bilayer in an unstructu...