PREDICTING THE EXPRESSION AND SOLUBILITY OF MEMBRANE PROTEINS

1

PREDICTING THE EXPRESSION AND SOLUBILITY OF
MEMBRANE PROTEINS Center for High Throughput
Structural Biology Mark E. Dumont, Michael
A. White, Kathy Clark, Elizabeth J. Grayhack,
and Eric. M. Phizicky Departments of
Biochemistry and Biophysics, and Pediatrics,
University of Rochester Medical Center,
Rochester, NY 14642

Summary The challenge of overexpression
and solubilization of eukaryotic integral
membrane proteins is one of the most significant
obstacles to structure determination of this
important class of proteins. To identify
properties of membrane proteins that may be
predictive of successful overexpression, we
analyzed expression levels of the genomic
complement of over 1,000 predicted membrane
proteins in a recently completed Saccharomyces
cerevisiae protein expression library.1 We
detected statistically significant positive and
negative correlations between high membrane
protein expression and protein properties such as
size, overall protein hydrophobicity, number of
transmembrane helices, and amino acid composition
of transmembrane segments. Expression levels of
membrane and soluble proteins exhibited a nearly
identical negative correlation with protein size
and the overall hydrophobicity. However,
high-level membrane protein expression was
positively correlated with the hydrophobicity of
predicted transmembrane segments. To further
characterize yeast membrane proteins as potential
targets for structure determination, we tested
the solubility of 123 of the highest expressed
yeast membrane proteins in six commonly used
detergents. Over 75 of our test proteins could
be classified into just four detergent solubility
patterns. Protein size, number of transmembrane
segments, and the hydrophobicity of predicted
transmembrane segments all showed significant
correlations with solubility in some detergents.
These results suggest that bioinformatic
approaches may be capable of identifying certain
classes of membrane proteins most likely to be
amenable to high level recombinant expression and
efficient detergent solubilization, facilitating
structural genomics approaches to membrane
protein structure determination. 1Gelperin DM,
White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ,
Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H,
Gerstein M, Dumont ME, Phizicky EM, Snyder
M, and Grayhack EJ. (2005) Genes Dev. 19,
2816-2826.
Testing Solubilization of High-Expressing Yeast
Proteins in Six Different Detergents
Solubilization Efficiency (123 total
proteins) Solid bars Effective
solubilization Hatched bars Partial
solubilization
Venn diagram of Proteins Solubilized by Different
Detergents
Detergents used Triton X-100 (TX-100),
lauryldimethylamine-N-oxide (LDAO), Fos-choline
12 (FC-12, dodecylphosphocholine),
tetraethyleneglycol monooctyl ether (C8E4),
n-octyl-b-D-glucoside (OG), and
n-dodecyl-b-D-maltoside (DDM). Procedure 9
?l of yeast whole-cell lysate (about 4.2 ?g
protein) were solubilized by addition of 141 ? l
of 1 TX-100, LDAO, FC-12, DDM, or 2 of OG and
C8E4 in 20 mM Hepes pH 7.5, 500 mM NaCl, and 10
glycerol, followed by centrifugation at 109,000
? g for 1 hour at 21oC. A portion of the
supernatant was diluted in loading buffer for SDS
PAGE then analyzed by immunoblotting using
anti-HA antibodies.

• 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
• 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)

• The MORF Yeast Protein Overexpression Library
• The yeast MORF library is a genomic collection of
  Saccharomyces cerevisiae strains expressing
  C-terminally tagged proteins under Gal control1
• The MORF library contains 5,574 sequence-verified
  clones tested for protein expression by Western
  blot

Detergent solubilization of membrane
proteins Correlations with number and polarity
of TM segments

• 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

Percent charged and polar residues in TM segments
Number of TM segments
u OG n C8E4
u OG n C8E4
p DDM l TX-100
p DDM l TX-100
MORF library vector insert region (Gateway
cloning)
Prediction of Yeast Transmembrane Proteins
Two different transmembrane helix prediction
programs were used to identify and classify
membrane proteins in the yeast genome. We used
TMHMM v. 2.02, http//www.cbs.dtu.dk/services/TMHM
M/) to predict 1,155 integral membrane proteins
in the MORF collection. From this set of 1,155
proteins, we removed 63 that were predicted by
the Phobius program3 (http//phobius.binf.ku.dk/)
to have only a signal peptide and no
transmembrane segments. This left a total of
1,092 proteins predicted to have one or more
transmembrane helices. Since TMHMM may not be
best for determining the actual topology of a
membrane protein4, we used HMMTOP predictions5
http//www.enzim.hu/hmmtop/) to predict of the
topology of the membrane proteins identified as
such by TMHMM. In very few cases where we were
aware of good experimental data suggesting a
topology different from the HMMTOP prediction, we
used this experimentally determined topology in
our analysis. 2Krogh A, Larsson
B, von Heijne G, and Sonnhammer EL. (2001) J Mol
Biol. 305567-80. 3Kall L, Krogh A, and
Sonnhammer EL. (2004) J Mol Biol. 3381027-36.
4Lehnert U, Xia Y, Royce TE, Goh CS, Liu Y,
Senes A, Yu H, Zhang ZL, Engelman DM, and
Gerstein M.(2004)Q. Rev Biophys. 37121-46.
5Tusnady GE, and Simon I.(2001) Bioinformatics
17849-50.
Conclusions 1. Membrane proteins can be
overexpressed on a genomic scale, many of them at
high levels. 2. Many factors affect
overexpression of soluble and membrane proteins
similarly. 3. While overall hydrophobicity of
membrane proteins is negatively correlated with
expression, hydrophobicity of membrane 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. Solubility in shorter
chain detergents is dependent on specific protein
properties. 7. Increasing polarity of protein TM
segments tends to decrease efficiency of
solubilization by short chain detergents.
Disagreement between TMHMM and HMMTop in
predictions of Transmembrane Proteins in the
Yeast Proteome