Lecture 33 Membrane Proteins

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1 Lecture 33 Membrane Proteins Reading for today: Chapter 4, section D Required reading for next Wednesday: Chapter 14, sections A and to the end Kuriyan, J., and Eisenberg, D. (2007) The origin of protein interactions and allostery in colocalization. Nature 450: Optional reading for Wednesday listed on the syllabus 4/22/16 1

2 Pop Question 11 Determine approximately how long it would take (a) a 30 kda protein and (b) a synaptic vesicle to diffuse 100 microns. For each case, compare spreading in simple aqueous buffer like you might use in lab vs. cytosol. r rmsd = (6Dt) 1/2 cytosol = 10 cp buffer = 1 cp r rmsd = 10 4 m t = r rmsd2 / 6D D = k B T / 6 r Part B: synaptic vesicle in cytosol (r = 20x10 9 m): D cytosol = (1.38x10 23 kg m 2 s 2 K 1 )(300 K) = 1.1x10 12 m 2 s 1 6(3.14)(0.01 kg m 1 s 1 )(2x10 8 m) t cytosol = ( m) 2 / 6(1.1x10 12 m 2 s 1 ) = 1,515 s or 25 min 1 In buffer: t buffer = t cytosol ( buffer ) / cytosol = 2.5 min 4/22/16 2

3 Pop Question 11 Determine approximately how long it would take (a) a 30 kda protein and (b) a synaptic vesicle to diffuse 100 microns. For each case, compare spreading in simple aqueous buffer like you might use in lab vs. cytosol. r rmsd = (6Dt) 1/2 cytosol = 10 cp buffer = 1 cp r rmsd = 10 4 m t = r rmsd2 / 6D D = k B T/ 6 r Part A: a 30 kda protein r MW 4 3( (1. 35 g ) / g/cm )( ) In cytosol: D cytosol = k B T/ 6 r = 1.1x10 7 m 2 s 1 t cytosol = (0.01 cm) 2 / 6 (1.1x10 7 cm 2 s 1 ) = 152 s or 2.5 min cm In buffer: D buffer = D cytosol ( cytosol ) / buffer t buffer = t cytosol ( buffer ) / cytosol = 2.5 min (1 cp) / 10 cp = 15 s 4/22/16 3

4 Today s Goals Membrane protein structures and how they compare to soluble proteins Hydropathy plots Distribution of different residue types on membrane protein structures Consequences in sequence analyses Membrane protein structure and function Features of ion channels vs. transporters Gates and selectivity of ion channels Alternating access of transporters Structural features of membrane proteins Breaks in helices serving as active sites Glycines and prolines as hinges Symmetry and pseudo symmetry 4/22/16 4

5 Identifying helical membrane proteins Helical membrane proteins have long stretches (~20 residues) of hydrophobic amino acids hydrophobic acidic basic polar Which type of membrane protein (α helical or β barrel) is easiest to identify by sequence alone? Figure from The Molecules of Life ( Garland Science 2008) 4/22/16 5

6 Kyte Doolittle hydrophobicity scale Kyte Doolittle hydrophobicity scale is one of the earliest scales and still widely used Values from combination of experimental, theoretical and sequence analysis data Kyte-Doolittle Hydrophobicity Scale 6 4 Charged Polar Hydrophobic Arg Lys Glu Asp Gln Asn His Pro Tyr Trp Ser Thr Gly Ala Met Cys Phe Leu Val Ile 4/22/16 6

7 Wimley White hydrophobicity scale Experimentally determined partitioning into a bilayer vs. water Determined using peptides with substituted side chains 4/22/16 7

8 Hydropathy plot Hydropathy plots use a sliding window (usually 19 residues) to calculate the average local hydrophobicity:...klrtplnyillnlavadlfmvlggftstlytslhgyfvfgptgcnlegffatlggeialw SLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWSRYIPEGLQCSCGI DYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTVKEAAAQQ... average? Out In Plot for bovine rhodopsin 4/22/16 8

9 Myoglobin hydrophathy plot Human myoglobin sequence: GLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILKK KGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG Window size = 19, threshold for possible transmembrane segments 4/22/16 9

10 Hydropathy plot for porin barrels don t have regions with hydrophobicity > 1.8 Out In 4/22/16 10

11 Some concepts to remember 2D diffusion measurements demonstrate the fluidity of biological membranes Biological membranes are bilayers of diverse lipids stabilized by the hydrophobic effect Biological membranes are organized in lipid rafts and microdomains Detergents can form micelles and substitute for membrane lipids to solubilize membrane proteins Biological membranes are bilayers of diverse lipids stabilized by the hydrophobic effect Membrane proteins have helices or barrels Hydropathy plots can identify helical transmembrane segments 4/22/16 11

12 Case study: hydropathy plots and protein purification You are interested in the structure and function of a novel protein. After cloning this protein and obtaining the predicted protein sequence, you create a hydropathy plot with window size of 19 residues, shown below: To purify the protein from cells, you screen for optimal lysis conditions by treating cells with different conditions, centrifuging to remove insoluble debris, and analyzing the supernatant by Western blotting: 1: Whole cells 2: Soluble fraction after hypotonic lysis 3: Soluble fraction after freeze and thaw 4: Soluble fraction after lysis in detergent From all of this information, make a reasonable hypothesis about the overall structure of the protein. 4/22/16 12

13 Case study: hydropathy plots and protein purification Hydropathy plot Western blotting 1: Whole cells 2: Soluble fraction after hypotonic lysis 3: Soluble fraction after freeze and thaw 4: Soluble fraction after lysis in detergent Cell lysis experiments indicate that it could be a membrane protein (soluble only in the presence of detergent), though it could also be an unstable soluble protein which is solubilized by detergent Hydropathy plot indicates that, if it is a membrane protein, it will be a β barrel, peripheral or lipid anchored membrane protein as no peaks of high hydropathy scores are observed 4/22/16 13

14 Aromatic residues at the interface Rhodopsin Porin Out Out In In Tyr and Trp, particularly, form collars at the lipid water interface Phe (grey on rhodopsin) distributes with the lipid 4/22/16 14

15 Arg and Lys snorkel to the surface Positively charged residues are favored near the negatively charged phosphate moieties of the phospholipids Out They snorkel to the interface, with their hydrophobic side chain portion interacting with the hydrophobic fatty acids Positive inside rule : higher density of positively charged residues on the intracellular face In 4/22/16 15

16 Hydrophobic residues in the hydrophobic region of the bilayer Aromatic residues particularly Tyr and Trp favor the lipid water interface (head group region) Long Arg and Lys side chains snorkel to the water surface, emphasizing their dual nature hydrophobic chain with charged terminus Killian and von Heijne (2000) TIBS 4/22/16 16

17 Membrane protein sequence alignments Membrane proteins have characteristic sequence features (e.g. more hydrophobic residues) Tools designed for sequence analyses of soluble proteins may not be optimal for membrane proteins Example: statistics based amino acid substitution matrices Their statistics are based on soluble protein amino acid composition Reminder: BLOSUM62 matrix substitution matrix describing the likelihood of finding a particular substitution in homologous proteins in nature 4/22/16 17

18 Amino acid substitution matrix BLOSUM62 Figure from The Molecules of Life ( Garland Science 2008) 4/22/16 18

19 BLOSUM62 how it was built Developed using a large dataset of sequence alignment blocks containing sequences that are clearly evolutionarily related Key variable: How often a particular pair of amino acids (e.g. F and L) occurs at a given position in the alignment Normalization factor: Relative abundance of the different amino acids How often the particular pairing would occur by random chance 4/22/16 19

20 BLOSUM62 matrix BLOSUM62 score: s ij = 2 log 2 L ij Log-based score makes it additive over alignment likelihood of i,j substitution observed frequency probability of random occurence (number of occurence of i,j pairs in same column total number of pairs 2n i n j 2 positions in the alignment) If the frequency of amino acid residues in the protein sequence of interest is different (e.g. more hydrophobic residues), then the matrix may not work as well Matrices have been generated specifically for the analysis of transmembrane proteins 4/22/16 20

21 PHAT75/73 is a membrane protein specific substitution matrix The PHAT (predicted hydrophobic and transmembrane) matrix is built using blocks of hydrophobic and transmembrane sequence segments. PHAT75/73 BLOSUM62 4/22/16 21

22 Cellular roles of membrane proteins Membrane proteins are the doors and windows of cells: Doors to let stuff in and out Windows to let cells sense their environment Extracellular milieu Neighboring cells Environment 4/22/16 22

23 Gatekeepers: Channels vs. Transporters Channel Transporter Selectivity filter Flow direction depends on electrochemical gradient Binding site(s) Unidirectional flow 4/22/16 23

24 Crossing cellular membranes: Channels vs. Transporters Channels allow molecules to cross membranes down an electrochemical gradient Selective pore Only requires a single gate No additional energy required for membrane crossing Fast ( ions per second, near diffusion limit) Transporters use an energy source to transport molecules across membranes, often against a concentration gradient Selective substrate binding site Requires two gates Slow (~30 50 per second) 4/22/16 24

25 Bacterial potassium channels KcsA a voltage and ph gated potassium channel MthK a calcium activated potassium channel Specific for potassium (~1.4 Å) vs. sodium (~1.0 Å) Formed of 4 identical subunits with 4 fold symmetry PyMOL KcsA structure 4/22/16 25

26 Some lessons from KcsA structure Not all helices span the membrane completely Resulting loops and unpaired hydrogen bonding groups are important for activity Many of the important groups in KcsA are actually main chain carbonyls The presence and importance of those groups was not discovered until the structure was determined because they cannot be mutated Membrane proteins can have cavities that are solvent accessible 4/22/16 26

27 KcsA vs. MthK channel gating Potassium channels open through a glycine hinged gate Out In MthK s C-terminal helix is kinked 4/22/16 27

28 Glycines as flexible hinges A glycine residue forms a flexible hinge that acts as the gate in potassium channels Conserved from bacteria tohumans Jiang (2002) Nature 4/22/16 28

29 Potassium channel gating movies View from inside the cell View from the side (Jiang et al (2002) Nature; supplemental information) 4/22/16 29

30 Crossing cellular membranes: Channels vs. Transporters Channels allow molecules to cross membranes down an electrochemical gradient Selective pore Only requires a single gate No additional energy required for membrane crossing Fast ( ions per second, near diffusion limit) Transporters use an energy source to transport molecules across membranes, often against a concentration gradient Selective substrate binding site Requires two gates Slow (~30 50 per second) 4/22/16 30

31 Transporters One or both steps require an expense of energy *** Two gates allow alternating access *** 4/22/16 31

32 Alternating access conformational change View from the top Two-fold axis perpendicular to the membrane One mechanism is based on a dimeric structure (either two identical or two homologous subunits) 4/22/16 32

33 Alternating access symporter Here, proton gradient is used to co transport a metal ion Binding of both substrates triggers a conformational change ordered sequential mechanism in H + Me H + Me H + out in Me Me H + out Me Many variations on this theme H + 4/22/16 33

34 Alternating access antiporter Here, ion gradient is used to transport a substrate, but the ions flow in the reverse direction compared to the substrate Mechanism? + S + S + S + S 4/22/16 34

35 Alternating access ATP driven In a substrate export mechanism: ATP binding promotes closure if transport substrate is bound ATP hydrolysis promotes opening and lowers the affinity for transport substrate, promoting release Out In Procko et al (2009) FASEB J 4/22/16 35

36 Alternating access second mechanism Two homologous halves Pseudo two fold in the plane of the membrane ~180 4/22/16 36

37 Alternating access another way Two fold in the plane of the membrane Repeat 1 Repeat 2 Repeat 1 Repeat 2 Repeat 1 is straight Repeat 2 is bent Repeat 1 is bent Repeat 2 is straight 4/22/16 37

38 Leucine transporter LeuT Bacterial leucine transporter homologue of neurotransmitter transporters Leu/Na + symporter pseudo 2 fold in the plane of the membrane Repeats 1 and 2 related by a 2- fold axis pointing out of the page Repeat 1 Repeat Yamashita et al (2005) Nature 4/22/16 38

39 LeuT structure Discontinuous helices form the substrate binding site In the middle of the membrane Out In Screpanti and Hunte (2007) J Structural Biology 4/22/16 39

40 Conformational change in LeuT Leu + Na + Leu + Na + Forrest L. R. et.al. PNAS 2008;105: /22/16 40

41 Aspartate transporter Bacterial homologue of the glutamate transporter in synapses Transporter conformational change How can there be so much sliding within the protein structure? Reyes et al (2009) Nature 4/22/16 41

42 Aspartate transport mechanism Reyes et al (2009) Nature 4/22/16 42

43 Some concepts to remember Aromatics are found at the lipid water interface, positively charged residues snorkel up to the phosphate groups of lipids Specialized scoring matrices take into account altered amino acid frequencies for sequence alignments Transmembrane helices pack tightly using knobs into holes or ridges into grooves patterns Some helices have breaks or do not span the whole membrane Functionally important motifs, active site locations Channels have a gate and a selectivity filter Ions move passively (through diffusion) when the gate is open Transporters use different alternating access mechanisms Need a source of energy to select and transport the substrate Glycines and prolines in helices can serve as hinges for conformational changes 4/22/16 43