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The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359-E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a "primed" open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182-17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications.

Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature 427:36-44; Zimmer J, et al. (2008) Nature 455:936-943; Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107:17182-17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid's contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal/(mol · Å(2)), the solvation parameter for transfer from translocon to bilayer is 6-10 cal/(mol · Å(2)), pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon.

The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (L(n), n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-L(n)-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n = 12. For X = K, p is already close to 100% for n = 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.

Transmembrane alpha-helices in integral membrane proteins can have two orientations in the membrane: N(in)-C(out) or N(out)-C(in). Previous studies of model N(out)-C(in) transmembrane segment have led to a detailed, quantitative picture of the "molecular code" that relates amino acid sequence to membrane insertion efficiency in vivo [Hessa T, et al. (2007) Molecular code for transmembrane helix recognition by the Sec61 translocon. Nature 450:1026-1030], but whether the same code applies also to N(in)-C(out) transmembrane helices is unknown. Here, we show that the contributions of individual amino acids to the overall efficiency of membrane insertion are similar for the two kinds of helices and that the threshold hydrophobicity for membrane insertion can be up to approximately 1 kcal/mol lower for N(in)-C(out) compared with N(out)-C(in) transmembrane helices, depending on the neighboring helices.

Voltage-sensitive ion channels open and close in response to changes in transmembrane (TM) potential caused by the motion of the S4 voltage sensors. These sensors are alpha-helices that include four or more positively charged amino acids, most commonly arginine. The so-called paddle model, based on the high-resolution structure of the KvAP K+ channel [Jiang, et al. (2003) Nature 423, 33-41], posits that the S4 sensors move within the membrane bilayer in response to TM voltage changes. Direct exposure of S4 sensors to lipid is contrary to the classical expectation that the dielectric contrast between the membrane hydrocarbon core and water presents an insurmountable energetic penalty to burial of electric charges. Nevertheless, recent experiments have shown that a helix with the sequence of KvAP S4 can be inserted across the endoplasmic reticulum membrane. To reconcile this result with the classical energetics argument, we have carried out a molecular dynamics simulation of an isolated TM S4 helix in a lipid bilayer. The simulation reveals a stabilizing hydrogen-bonded network of water and lipid phosphates around the arginines that reduces the effective thickness of the bilayer hydrocarbon core to approximately 10 A in the vicinity of the helix. It suggests that bilayer phospholipids can adapt locally to strongly perturbing protein elements, causing the phospholipids to become a structural extension of the protein.

The solvation energies of salt bridges formed between the terminal carboxyl of the host pentapeptide AcWL- X-LL and the side chains of Arg or Lys in the guest (X) position have been measured. The energies were derived from octanol-to-buffer transfer free energies determined between pH 1 and pH 9. 13C NMR measurements show that the salt bridges form in the octanol phase, but not in the buffer phase, when the side chains and the terminal carboxyl group are charged. The free energy of salt-bridge formation in octanol is approximately -4 kcal/mol (1 cal = 4.184 J), which is equal to or slightly larger than the sum of the solvation energies of noninteracting pairs of charged side chains. This is about one-half the free energy that would result from replacing a charge pair in octanol with a pair of hydrophobic residues of moderate size. Therefore, salt bridging in octanol can change the favorable aqueous solvation energy of a pair of oppositely charged residues to neutral or slightly unfavorable but cannot provide the same free energy decrease as hydrophobic residues. This is consistent with recent computational and experimental studies of protein stability.

Tanford [Tanford, C. (1979) Proc. Natl. Acad. Sci. USA 76, 3318-3319] used thermodynamic arguments to show that the pressure difference across the bilayer of small phospholipid vesicles must be zero. This paper analyzes the implications of this conclusion in terms of Laplace's law and the basic thermodynamics of interfaces. In its usual form, Laplace's law is of questionable value for the vesicle. If the vesicle is in a state of metastable equilibrium, the surface free energy must be minimal with respect to several thermodynamic variables; the condition (delta F/ delta A) = 0 is not adequate by itself.

Knowledge of the molecular packing of lipids and water in lipid bilayers is important for understanding bilayer mechanics and thermodynamics. Information on packing is most often obtained from x-ray or neutron diffraction measurements. Given the d spacing, composition, and partial specific volumes of the lipid and water, it is a simple matter to calculate the area per lipid molecule, bilayer thickness, and bilayer mass density. The partial specific volumes are commonly assumed to be those of bulk water and of lipid in excess water regardless of the degree of bilayer hydration. We present evidence here that these assumptions should be seriously questioned. At low hydrations, we find the head groups of egg and dioleoyl lecithin to be much less tightly packed than previously thought and the partial specific volume of water to be considerably smaller than 1 ml/g. Because the molecular packing affects the mechanical properties of bilayers, we use the results to reevaluate published experiments concerning the elastic area compressibility modulus of egg lecithin bilayers and the repulsive hydration force between bilayers.