PREDICTING THE EXPRESSION AND SOLUBILITY OF MEMBRANE PROTEINS
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1 PREDICTING THE EXPRESSION AND SOLUBILITY OF MEMBRANE PROTEINS Mark E. Dumont, Michael A. White, Kathy Clark, Elizabeth J. Grayhack, and Eric. M. Phizicky
2 Unknowns in Membrane Protein Expression, and Solubilization ( + Crystallization) 1. Criteria for choice of membrane protein targets resulting in need to test protein variants and related proteins from many species. - Lack of fundamental understanding of factors governing expression, solubilization, crystallization 2. Choice of detergent for solubilization- usually performed by trial and error - Lack of fundamental understanding of physical-chemical basis of membrane protein interactions with detergents.
3 The MORF Yeast Protein Overexpression Library A genomic collection of S. cerevisiae strains over-expressing C- terminally tagged proteins under Gal control - 5,574 sequence-verified clones tested for protein expression by Western blot - A joint project of the laboratories of: Mark Dumont Elizabeth Grayhack (University of Rochester) Eric Phizicky Michael Snyder (Yale University) MORF library vector insert region (Gateway cloning) P GAL ATT site ORF ATT site His6 HA 3C ZZ Gelperin et al., Genes & Development 19(23), Dec 1, 2005
4 The Problem of Identifying Transmembrane Proteins Disagreement between TMHMM and HMMTop in predictions of Transmembrane Proteins in the Yeast Proteome Total TM ORFs (HMMTop) 2037 Total TM ORFs (TMHMM) 1168 ORFs where predictions disagree: 1375
5 Widespread Expression of Membrane Proteins in the MORF Library. - 95% of cloned soluble proteins in the MORF library are expressed. - 88% of cloned predicted membrane proteins in the MORF library are expressed Membrane Proteins All MORF Proteins Gelperin et al., Genes & Development 19(23), Dec 1, 2005
6 How do high-expressing membrane proteins differ from low expressing ones? Factors evaluated for correlations with membrane protein expression and solubilization: Codon usage, codon adaptation index Molecules per cell under chromosomal expression Percentage of total protein residues that are aromatic Isoelectric point Size (kda) GRAVY score (overall protein hydrophobicity) Homolog in yeast or other organism Cellular membrane localization Predicted to contain a signal peptide Membrane protein characteristics Number of predicted transmembrane segments N- and C-terminal orientation across membrane Average transmembrane segment length Percentage of protein in transmembrane segments Percentage of transmembrane residues that are hydrophobic (WFLIVMY) Percentage of transmembrane residues that are charged/polar (EDKRHNQST) Percentage of transmembrane residues that are aromatic (WYF)
7 Protein size and overall hydrophobicity are negatively correlated with expression levels of soluble and membrane proteins Size range (kda) Overall hydrophobicity (GRAVY Score) p = 2.5x10-5 Soluble proteins Membrane proteins p = 4.1x10-9
8 Expression of membrane proteins is positively correlated with the hydrophobicity of TM segments Percent charged and polar residues in TM segments Percent hydrophobic residues in TM segments p = 3.1x10-9 p = 6.0x10-4 = Membrane proteins Bars = Number of ORFs per bin
9 Factors Not Significantly Associated with Expression N- and C- terminal orientations Subcellular localization Presence of predicted signal peptide
10 Detergent Solubilization of High-Expressing Yeast Membrane Proteins 263 predicted integral membrane proteins in MORF expressed ~1mg/L 90 have human orthologs >40 with activities compatible with published assays
11 Detergents used for solubilization C8E4 Octyl-glucoside LDAO Fos-choline 12 Dodecyl-maltoside TX-100
12 Examining Solubility by Western Blot
13 Solubilization Efficiency (123 total proteins) Effective solubilization Partial solubilization
14 Venn Diagram of Proteins Solubilized by Different Detergents
15 Protein Characteristics Associated with Solubility in Short-Chain Detergents Number of TM segments Percent charged and polar residues in TM segments DDM TX-100 OG C8E4 DDM TX-100 OG C8E4 OG: p = 1.2 x10-2 C8E4: p = 2.6 x10-4 OG: p = 2.1 x10-2 C8E4: p = 2.1 x10-2
16 Nonly Octyl Heptyl No detergent Lysate Decyl Nonly Octyl Heptyl No detergent Lysate Decyl Nonly Octyl Heptyl No detergent Lysate Acyl chain length dependence of solubilities in detergents of the acyl-maltoside family Cho1p Ost6p Sec66p Decyl
17 Conclusions 1. Membrane proteins can be overexpressed on a genomic scale, many of them at high levels. 2. Some factors (size, pi, native expression level) affect overexpression of soluble and membrane proteins similarly. 3. Overall hydrophobicity of membrane proteins is negatively correlated with expression but hydrophobicity of TM regions is positively correlated with expression. 4. The presence of a predicted signal sequence, topological orientation in the membrane, and normal subcellular localization do not appear to affect the ability of yeast membrane proteins to be overexpressed. 5. The majority of yeast membrane proteins can be solubilized using a small set of detergents. 6. Increasing polarity of protein TM segments tends to decrease efficiency of solubilization by short chain detergents.
18 Michael White Also: Kathy Clark Elizabeth Grayhack Eric Phizicky
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