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[show abstract]
[hide abstract]
ABSTRACT: Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism
whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy
assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two
AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic
and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic
interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive
binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the
sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane
but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish
within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest
that the description of membrane curvature-sensing requires consideration of several factors such as short and long range
electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.
Journal of Biological Chemistry 12/2011; 286(49):42603-42614. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues.
Journal of Biological Chemistry 09/2011; 286(49):42603-14. · 4.77 Impact Factor
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[hide abstract]
ABSTRACT: In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding sequence allowed tuning of protein expression over ~300-fold with preservation of amino acid identity. This approach is simple and should be generally applicable in bacteria. The data support that features in the 5' mRNA coding region near the AUG start codon are key in determining translation output and hence is important to recombinant and, most certainly, endogenous gene expression.
Protein Engineering Design and Selection 11/2010; 24(1-2):123-9. · 2.94 Impact Factor
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Jobin Varkey,
Jose Mario Isas,
Naoko Mizuno, Martin Borch Jensen,
Vikram Kjøller Bhatia,
Christine C Jao,
Jitka Petrlova,
John C Voss,
Dimitrios G Stamou,
Alasdair C Steven,
Ralf Langen
[show abstract]
[hide abstract]
ABSTRACT: Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.
Journal of Biological Chemistry 10/2010; 285(42):32486-93. · 4.77 Impact Factor
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Jobin Varkey,
Jose Mario Isas,
Naoko Mizuno, Martin Borch Jensen,
Vikram Kjøller Bhatia,
Christine C. Jao,
Jitka Petrlova,
John C. Voss,
Dimitrios G. Stamou,
Alasdair C. Steven,
Ralf Langen
[show abstract]
[hide abstract]
ABSTRACT: Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction
for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that
synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here,
we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature
and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically
similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however,
synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane
insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures
that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees
well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane
curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction
depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be
important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease
remains to be determined.
Journal of Biological Chemistry 10/2010; 285(42):32486-32493. · 4.77 Impact Factor
