1. Hydrophobic Mismatch between Proteins and Lipids in Membranes Susanne Pfeifer tiffy@tiffy.it 08.07.2004 Seminar Theoretical Analysis of Protein-Protein Interactions Universität des Saarlandes Chair of Prof. Dr. Volkhard Helms

2. 2 Agenda Introduction Possible adaptations to mismatch Consequences of mismatch for: Proteins and peptides Lipid structure and organization Effects of mismatch in biomembranes

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4. 4 Basics Introduction

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6. 6 Length of lipid-exposed hydrophobic segments is equal to the hydrophobic bilayer thickness Proteins that are encountered in one membrane can have different lengths of their hydrophobic parts Membrane proteins with the same length can be encountered in bilayers of different thickness

7. 7 Basics Questions 1.How do membranes deal with a mismatch between the hydrophobic part of a transmembrane protein and the bilayer thickness? 2.How important is the extent of hydrophobic matching for membrane structure and function? 3.Could mismatch play a functional role?

8. 8 Basics Possible adaptations to mismatch Positive mismatch The protein might oligomerize or aggregate in the membrane to minimize the exposed hydrophobic area Transmembrane helices could tilt to reduce their effective hydrophobic length Transmembrane helices could adopt another conformation

9. 9 Basics Possible adaptations to mismatch Negative mismatch Results in protein aggregation or changes in backbone conformation or side chain orientation Too short peptides might not incorporate and adopt a surface localization Lipids decrease the bilayer thickness by disordering their acyl chains

10. 10 Basics Implications for membranes Effects on protein conformation protein orientation helical tilt aggregational behavior can affect protein activity membrane insertion protein assembly Effects on lipid structure lipid organization have implications for processes that are sensitive to lipid packing Processes that require the local and transient formation of non-lamellar structures

11. 11 Basics Consequences of mismatch Consequences for properties of proteins Protein activity and stability Protein aggregation Tilt Localization at membrane surface Protein/peptide backbone conformation

12. 12 Basics - Consequences of mismatch Protein activity & stability The extent of hydrophobic matching is important for determining the functional activity of proteins There are a number of proteins that do not show a clear optimum bilayer thickness for activity, but they require a minimal chain length many other factors may be involved in determining the functional activity of membrane proteins (e.g. lipid packing, fluidity, surface charge density, intrinsic curvature, lateral pressure profile, …)  Protein activity may be related to protein stability, which also can be affected by mismatch

13. 13 Basics - Consequences of mismatch Protein aggregation Response to hydrophobic mismatch Occurred only with a rather large mismatch: 4 Å thicker or 10 Å thinner than the estimated hydrophobic length of the protein are allowed without induction of significant aggregation Proteins with long hydrophobic stretch tilt in the membrane  Reduction of their effective length Comparison is difficult, because the lipids differ not only in acyl chain length, but also in other properties

14. 14 Basics - Consequences of mismatch Tilt Occurs if the hydrophobic part of a protein is too long to span the membrane Important for the functional and transport activity of membrane proteins An increase in helix tilt occurs at increasing protein content  decrease in lipid order  decrease in bilayer thickness Accompanied by a bend to reduce unfavorable effects on lipid packing

15. 15 Basics - Consequences of mismatch Tilt Change in helix tilt change in protein activity

16. 16 Basics - Consequences of mismatch Tilt Special cases: In large proteins: changes in helical tilt have only little effect on lipid packing Single transmembrane helix: a tilt would cause a strain on the surrounding lipids to accommodate the helix in the bilayer  large degree of tilting is less favorable

17. 17 Basics - Consequences of mismatch Localization at membrane surface Relatively small hydrophobic peptides may not be able to integrate into the membrane orientation at the membrane surface Peptide aggregation outside the bilayer Amino acid composition is important (in determining the consequences of hydrophobic mismatch) The extent of membrane insertion for amphipathic pore-forming peptides is mismatch dependent

18. 18 Basics - Consequences of mismatch Localization at membrane surface Surface-absorbed peptides insert their hydrophobic side chains between the acyl chains near the membrane surface membrane-thinning effect dependent on the peptide/lipid ratio Important for studies on the mismatch dependence of insertion for such proteins insertion of hydrophobic peptides with an equilibrium between a transmembrane orientation and a surface localization

19. 19 Basics - Consequences of mismatch Backbone conformation Helix length fluctuates due to local variations in backbone structure Sensitivity of the backbone conformation for environmental changes depends on amino acid composition Peptides with a hydrophobic stretch of alternating leucine and alanine are more sensitive than peptides with a polyleucine sequence

20. 20 Basics Consequences of mismatch Consequences for lipid structure and organization Lipid chain order Phase transition temperature Preferential interactions and microdomain formation

21. 21 Basics - Consequences of mismatch Phase transition temperature Melting transition temperature of lipid bilayers is strongly affected Proteins with long hydrophobic segments stabilize the thicker gel phase Short proteins stabilize the fluid phase

22. 22 Basics - Consequences of mismatch Microdomains In fluid bilayers consisting of lipids with different lengths, hydrophobic mismatch may induce preferential protein-lipid interactions  formation of microdomains Systems consisting of two lipid species with different acyl chain lengths and one protein: hydrophobic mismatch induces preferential protein-lipid interactions (depending on hydrophobic length, differences in hydrophobic length…)

23. 23 Basics Effects in biomembranes Protein sorting Membrane protein insertion and topology Regulation

24. 24 Basics - Effects in biomembranes Protein sorting Eukaryotic cell: Level of cholesterol increases from the endoplasmatic reticulum via the Golgi to the plasma membrane (suggesting a concomitant increase in membrane thickness) Protein sorting in Golgi is based on this length difference Increasing the hydrophobic length of proteins that normally reside in the Golgi  they can reroute the proteins to the plasma membrane (or vice versa)

25. 25 Basics - Effects in biomembranes Protein sorting Preferential protein-lipid interactions are consequences of hydrophobic mismatch  results in domain formation and protein sorting

26. 26 Basics - Effects in biomembranes Membrane protein insertion Signal sequences: short hydrophobic length (7-15 amino acids) high tendency to form alpha-helical structures (with insufficient length to span a membrane) Length of signal sequences and mismatch are important for their functional activity A mismatch could lead to a local destabilization in a bilayer  helps the translocation or  promotes preferential interactions with other short helices of proteins in the translocation machinery

27. 27 Basics - Effects in biomembranes Membrane protein insertion Signal anchors length closer to the hydrophobic thickness of the membrane (19-27 amino acids)  influences the topology of proteins Stop transfer sequences  hydrophobicity is more important than length

28. 28 Basics - Effects in biomembranes Membrane thickness regulation A large variation in membrane thickness can be tolerated Variations of acyl chain length lead to changes in lipid composition important for surface charge density serves as tool to regulate local bilayer thickness  prevention of unwanted consequences of hydrophobic mismatch in biological membranes

29. 29 Basics Results Hydrophobic mismatch affects protein and lipid organisation affects conformation and thermodynamic properties of the membranes plays a role in protein sorting in vivo is required for specific functional properties of membranes depends on individual properties

30. 30 Chain Packing Calculation of all possible lipid conformations Probability of chain conformations relative to their distances Free interaction energy between two inclusions  Detailed molecular-level information on chain conformational properties Problems: Computationally expansive Full minimization of membrane shape is difficult

31. 31 Directors Model Theory-based model of elastic deformations is used to describe free energy differences associated with membrane perturbation due to protein-bilayer interactions (Huang, 1986; Helfrich and Jacobsson, 1990; Nielsen et. al. 1998) All parameters were used before in previous studies Thin, solvent-free lipid bilayer With an embedded inclusion similar to a gramicidin channel

32. 32 Directors Model - Theory The Model

33. 33 Directors Model - Theory The Model

34. 34 Directors Model - Theory Approximation of changes Elastic modes for approximation of changes in lipid packing: Compression-Expansion (CE) (due to changes in bilayer thickness) Splay-Distortion (SD) (due to variation in director among adjacent mol.) Surface-Tension (ST) (due to changes in bilayer surface area)

35. 35 Directors Model - Theory Total deformation free energy compression-expansion surface tension splay-distortion

36. 36 Directors Model - Results Choice of boundary conditions The bilayer deformation energy varies as a function of mechanical moduli boundary conditions Problem: Energetic costs for packing the lipid molecules which are adjacent to the inclusion are not considered!

37. 37 Directors Model - Results Choice of boundary conditions

38. 38 Directors Model - Results Bilayer deformation profile The shape of the deformation varies as a function of the elastic moduli Depending on the value of s, may the bilayer deformation profile be nonmonotonic  Energy minimization requirement may cause a compression adjacent to the inclusion and an expansion further away from the bilayer/inclusion boundery  Packing Problem hydrophobic core volume per unit bilayer surface will deviate from its equilibrium value

39. 39 Directors Model - Results Bilayer deformation profile

40. 40 Directors Model - Results Bilayer deformation profile

41. 41 Directors Model - Results Bilayer deformation profile

42. 42 Directors Model - Results Radial decomposition of free energy Depending on the choice of boundary conditions  G CE can be less, equal or larger than  G SD The relative contributions of these major components to  G def vary in dependence of s (contact slope)  (length scale)

43. 43 Directors Model - Results Radial decomposition of free energy

44. 44 Directors Model - Results Radial decomposition of free energy

45. 45 Directors Model - Discussion Comparison The results of the presented model confirm and extend the findings of Huang (1986) and Helfrich and Jakobsson (1990) Better results for s=0 Failures with s=s min could arise because the parameters that are used may be inappropriate or additional contributions to  G def which are neglected Today there is insufficient information to choose the appropriate boundary conditions

46. 46 Directors Model - Discussion Biological implications

47. 47 Appendix References Hydrophobic mismatch between proteins and lipids in membranes (1998, Killian) Energetics of Inclusion-Induced Bilayer Deformations (1998, Nielson et al) A Molecular Model for Lipid-Protein Interactions in Membranes: The Role of Hydrophobic Mismatch (1993, Deborah et al) Synthetic peptides as models for intrinsic membrane proteins (2003, Killian)

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