Review. Membrane proteins. Membrane transport

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1 Quiz 1

2 For problem set 11 Q1, you need the equation for the average lateral distance transversed (s) of a molecule in the membrane with respect to the diffusion constant (D) and time (t). s = (4 D t) 1/2 2

3 Review Membrane proteins Membrane transport 3

4 The Structure and Chemistry of Membrane Proteins Integral (intrinsic) proteins Peripheral (extrinsic) proteins lipid-anchored proteins 4

5 Integral Membrane Proteins are Imbedded in the Membrane Transmembrane region hydrophobic does not fold similarly to soluble proteins because of the hydrophobic lipids They can only be removed from the membrane by denaturing the membrane (organic solvents, or strong detergents) α-helical bundles found in plasma membrane of eukaryotes and inner membrane of bacteria, mitochondria, and chloroplasts β-barrel (even number of β-strands) located in outer membranes Glycophorin, bacteriorhodopsin are examples 5

6 Glyophorin A single-transmembrane-segment protein One transmembrane segment with globular domains on either end Transmembrane segment is alpha helical and consists of 19 hydrophobic amino acids Extracellular portion contains oligosaccharides and these constitute the ABO and MN blood group determinants 6

7 Bacteriorhodopsin A 7-transmembrane-segment (7-TMS) protein Found in purple patches of Halobacterium halobium Consists of 7 transmembrane helical segments with short loops that interconnect the helices br is a light-driven proton pump! transmembrane alphahelix requires residues 7

8 Porins Structure of maltoporin from E. coli. Found both in Gram-negative bacteria and in mitochondrial outer membrane Porins are pore-forming proteins kd General or specific Porin from Rhodobacter capsulatus has 16-stranded beta barrel that traverses the membrane to form the pore Beta-strand requires only 9-11 residues per transmembrane strand 8

9 Lipid-Anchored Membrane Proteins Four types have been found: Amide-linked myristoyl anchors Thioester-linked fatty acyl anchors Thioether-linked prenyl anchors Glycosyl phosphatidylinositol anchors 9

10 Membrane Transport what do you need to know? Read chapter 9 to p. 295 Facilitated diffusion, channels, active transport Equation of electrostatic potential (i.e. which direction will an ion move acording to chemical and electrical gradient) I will provide example problems in the next problem set Concept of primary and secondary transporters Note for problem set 11 ouabain is a potent inhibitor of the Na+- K+ pump 10

11 Interfaces between two molecules Size, shape, and charge of biomolecular complementarity surface area is a measure of the size of interaction Steric and electrostatic complementarity 11

12 Protein-DNA Interactions Transcription Translation DNA RNA protein DNA binding proteins have a central role in genetic activity transcription (trnascription factors) packaging (histones) rearrangement (topoisomerases) replication (polymerase) repair (thiamine dimer excision and repair) endonucleases (protection) 2-3 % of prokaryotic genome and 6-7 % of eukaryotic genome encodes DNA- binding proteins Additional Reading Chapter 29 pages

13 Protein-DNA Interactions We have mentioned basic residues interacting with the phosphate backbone as an interaction between proteins and DNA, but how is sequence specificity obtained? That is, the phosphate backbone is the same throughout the sequence; however, proteins bind to specific sequences, how? 13

14 e.g. TATA box binding protein The first eukaryotic genes to be sequenced and studied in in vitro transcription systems were viral genes and cellular protein-coding genes that are very actively transcribed either at particular times of the cell cycle or in specific differentiated cell types. In all of these rapidly transcribed genes, a highly conserved sequence called the TATA box was found base pairs upstream of the start site Stepwise assembly of a transcriptioninitiation complex starts with TATAbinding protein (TBP) and recruits RNA polymerase II (Pol II) and general transcription factors. 14

15 H-bond definition: H-A <2.7 Å, D-A distance <3.35 Å, D-H-A angle > 90 15

16 Schematics of bidentate interactions Arrow points from donor to acceptor 16

17 Schematics of bidentate interactions 17

18 Interactions with stacked bases 18

19 Interactions with diagonally-positioned bases 19

20 Van der Waals: all atoms not involved in H-bonds, but separated by < 3.9 Å 20

21 Spatial distribution of H-bonding Spatial distribution of van der Waals Major groove Minor groove 21

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23 HTH Motif 20 aa segment of two almost perpendicular α- helices Connected by a four residue turn Bind major grove sometime additional contact to the minor groove 23

24 Zinc-coordinating proteins Tetrahedral coordination of Zn by cys and his Predominantly major groove interactions 24

25 Zipper-type proteins Major groove interactions 25

26 Other α-helix proteins 26

27 β-sheet proteins Minor groove interactions! 27

28 β-hairpin/ribbon proteins 28

29 Protein Protein Interactions Handout PPI_I and PPI_II on Toolkit 29

30 What are protein protein interactions? The driving force for quaternary interactions Homooligomers Heterooligomers 30

31 Why are protein protein interactions important? 31

32 Distribution of the multimeric state of known protein structures 32

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34 Amino acid propensities at proteinprotein interfaces 34

35 Hot Spot Analysis Alanine scanning mutagenesis paired with measuring binding free energy of interaction indicates important residues for protein-protein interactions Trp, Arg, and Tyr common hot spot residues (this does not necessarily disagree with previous slide it is a different point) 35

36 Human growth hormone and receptor R W I W Numbering in PDB different I365, R243 36

37 ZipA and FtsZ interaction Complex is shown open Homework is to look at complementarity of both shape and hydrophobic interactions 37

38 Streptococcal protein G and human immunoglobin IgG I253 IgG S254 IgG Glu27 Protein G Hot spots on one protein surface Usually complement hot spots on the other protein 38

39 SP4206 Drug design cytokine receptor drug development IL-2Rα IL-2Rα SP4206 IL-2Rα SP

40 What do you need to know? Be able to identify and propose stabilizing interactions between biological interfaces based on the examples discussed today 40

41 Reading for Thursday The papers from the other Project II Chaperones translocon Chapter 31 Revisit Chapter 30 (protein-rna interactions in the ribosome) 41