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Biophysical studies of membrane associated peptides
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Astrid Gräslund)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A large part of the processes in living organisms involves proteins acting in a biological membrane. Biophysical studies on isolated model systems can give important understandings of the complicated biological mechanisms in the membrane. In this thesis peptide membrane interaction mechanisms are studied in several different systems. The membrane interactions of the unstructured endogenous opioid peptides dynorphinA (DynA) and dynorphinB (DynB) were investigated with Saturation Transfer Difference (STD) experiments, supplemented by various other NMR methods. The combined results support a conclusion that DynA binds to the lipid bilayer with the N-terminal residues inserted into the hydrophobic region and the C-terminal residues more loosely attached to the surface, while DynB is situated parallel to the bilayer. This difference in membrane interaction can explain observations that DynA has membrane perturbing effects while DynB has not. In the second study the binding domain of the glycosyltransferase A.laidlawii Monoglycosyldiacyl Glycerol Synthase (alMGS) was predicted and investigated mainly with NMR which enabled the determination of the 3D structure and position in a lipid environment. The phospholipid bilayers induce a large amphipathic α-helical content in the peptide, which aligns parallel but slightly tilted along the lipid surface with the N-terminus situated closer to the hydrophobic region. Lipid perturbation effects caused by peptide-membrane interactions were investigated by studying the influence of model transmembrane peptides on lipid dynamics in phospholipid bicelles with varying bilayer thickness. 13C-relaxation NMR of the lipids was used to survey the effects of the model peptides on the lipid bilayer.In paper IV and V structure and membrane interaction properties of the highly charged and flexible helix-turn-helix motif named the 'voltage sensor paddle' from two transmebrane voltage gated potassium channels was investigated. In membrane mimetic media, the KvaP paddle adopts the same type of helix-turn-helix conformation which can be seen in the Xray structure of the entire ion channel. The membrane interaction of the paddle HsapBK was compared with the corresponding one in KvaP, and both were inserted in the lipid bilayer but perturbed the lipid system differently, which may indicate differences in their function. Paper VI treats the structure of the novel site-specific fluorophore ReAsH bound to an optimized peptide sequence. The analysis shows that the important peptide mid segment configuration of CCPGCC is optimal for the ReAsH binding and that the N-terminal Phe1 plays an important role for the fluorophore process.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2009. , 55 p.
Keyword [en]
NMR, spectroscopy, membrane interaction, bicelles
National Category
Chemical Sciences
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-27488ISBN: 978-91-7155-888-6 (print)OAI: oai:DiVA.org:su-27488DiVA: diva2:214487
Public defence
2009-06-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2009-05-14 Created: 2009-05-05 Last updated: 2012-08-28Bibliographically approved
List of papers
1. Biophysical studies of the membrane location of the voltage-gated sensors in HsapBK and KvAP
Open this publication in new window or tab >>Biophysical studies of the membrane location of the voltage-gated sensors in HsapBK and KvAP
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-24834 (URN)
Note
Part of urn:nbn:se:su:diva-7488Available from: 2008-05-08 Created: 2008-05-08 Last updated: 2010-01-14Bibliographically approved
2. Membrane Interactions of Dynorphins
Open this publication in new window or tab >>Membrane Interactions of Dynorphins
2006 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, 15931-15940 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-19182 (URN)10.1021/bi061199g (DOI)
Available from: 2007-10-22 Created: 2007-10-22 Last updated: 2017-12-13Bibliographically approved
3. Lipid dynamics in fast-tumbling bicelles with varying bilayer thickness: Effect of model transmembrane peptides
Open this publication in new window or tab >>Lipid dynamics in fast-tumbling bicelles with varying bilayer thickness: Effect of model transmembrane peptides
2008 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, no 11, 2526-2534 p.Article in journal (Refereed) Published
Abstract [en]

The morphology of q=0.5 fast-tumbling bicelles prepared with three different acyl chain lengths has been investigated by NMR. It is shown that bicelles prepared with DLPC (12 C) and DHPC are on average larger than those containing DMPC or DPPC (14 and 16 C) and DHPC, which may be due to a higher degree of mixing between DLPC and DHPC. The fast internal mobility of the lipids was determined from natural abundance carbon-13 relaxation. A similar dynamical behaviour of the phospholipids in the three different bicelles was observed, although the DPPC lipid acyl chain displayed a somewhat lower degree of mobility, as evidenced by higher generalized order parameters throughout the acyl chain. Carbon-13 relaxation was also used to determine the effect of different model transmembrane peptides, with flanking Lys residues, on the lipid dynamics in the three different bicelles. All peptides had the effect of increasing the order parameters for the DLPC lipid, while no effect was observed on the longer lipid chains. This effect may be explained by a mismatch between the hydrophobic length of the peptides and the DLPC lipid acyl chain.

Identifiers
urn:nbn:se:su:diva-14858 (URN)000261263500009 ()18692021 (PubMedID)
Available from: 2008-11-05 Created: 2008-11-05 Last updated: 2017-12-13Bibliographically approved
4. High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase
Open this publication in new window or tab >>High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase
Show others...
2007 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 19, 5664-5677 p.Article in journal (Refereed) Published
Abstract [en]

In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.

Identifiers
urn:nbn:se:su:diva-24460 (URN)10.1021/bi700042x (DOI)000246283600005 ()
Note
Part of urn:nbn:se:su:diva-7056Available from: 2007-09-13 Created: 2007-09-04 Last updated: 2017-12-13Bibliographically approved
5. Hairpin Structure of a Biarsenical−Tetracysteine Motif Determined by NMR Spectroscopy
Open this publication in new window or tab >>Hairpin Structure of a Biarsenical−Tetracysteine Motif Determined by NMR Spectroscopy
Show others...
2009 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 13, 4613-4615 p.Article in journal (Refereed) Published
Abstract [en]

The biarsenical−tetracysteine motif is a useful tag for genetic labeling of proteins with small molecules in living cells. The present study concerns the structure of a 12 amino acid peptide FLNCCPGCCMEP bound to the fluorophore ReAsH based on resorufin. 1H NMR spectroscopy was used to determine the solution structure of the complex formed between the peptide and the ReAsH moiety. Structure calculations based on the NMR results showed that the backbone structure of the peptide is fairly well defined, with a hairpinlike turn, similar to a type-II β-turn, formed by the central CPGC segment. The most stable complex was formed when As2 was bonded to C4 and C5 and As1 to C8 and C9. Two clear NOESY cross-peaks between the Phe1 side chain and ReAsH confirmed the close positioning of the phenyl ring of Phe1 and ReAsH. Phe1 was found to have an edge−face geometry relative to ReAsH. The close interaction between Phe1 and ReAsH may be highly significant for the fluorescence properties of the ReAsH complex.

National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-27483 (URN)10.1021/ja809315x (DOI)000264806300030 ()
Available from: 2009-05-05 Created: 2009-05-05 Last updated: 2017-12-13Bibliographically approved
6. Solution structure of the HsapBK K+ channel voltage-sensor paddle sequence
Open this publication in new window or tab >>Solution structure of the HsapBK K+ channel voltage-sensor paddle sequence
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 25, 5813-5821 p.Article in journal (Refereed) Published
Abstract [en]

Voltage-gated potassium channels open and close in response to changes in the membrane potential. In this study, we have determined the NMR solution structure of the putative S3b-S4 voltage-sensor paddle fragment, the part that moves to mediate voltage gating, of the HsapBK potassium channel in dodecylphosphocholine (DPC) micelles. This paper presents the first structure of the S3b-S4 fragment from a BK channel. Diffusion coefficients as determined from PFG NMR experiments showed that a well-defined complex between the peptide and DPC molecules was formed. The structure reveals a helix-turn-helix motif, which is in agreement with crystal structures of other voltage-gated potassium channels, thus indicating that it is feasible to study the isolated fragment. The paddle motifs generally contain several basic residues, implicated in the gating. The critical Arg residues in this structure all reside on the surface, which is in agreement with crystal structures of K(v) channels. Similarities in the structure of the S3b-S4 fragment in BK and K(v) channels as well as important differences are seen, which may be important for explaining the details in paddle movement within a bilayer.

Keyword
NMR solution structure, S3b−S4 fragment, paddle
National Category
Biophysics
Research subject
Biophysics; Biochemistry
Identifiers
urn:nbn:se:su:diva-31727 (URN)10.1021/bi9004599 (DOI)000267326500006 ()19456106 (PubMedID)
Funder
Swedish Research Council, 621-2011-5964
Available from: 2009-11-25 Created: 2009-11-25 Last updated: 2017-12-12

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