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Membrane Induced Structure in Transmembrane Signaling Proteins and Peptides: Peptide–Lipid Interactions Studied by Spectroscopic Methods
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological membranes, defining the boundary of cells and eukaryotic organelles, are mainly composed of lipids and membrane proteins. Interactions between these lipids and proteins are needed to preserve the tight seal of the membrane, but also to induce structure for proper function in many membrane proteins. In this thesis, interactions between three different kinds of peptides, i.e. small proteins, and model membranes are studied by spectroscopic methods.

First, the membrane interaction of two paddle domains, KvAPp, from the voltage-gated potassium channel KvAP from Aeropyrum pernix, and HsapBKp, from the human, large conductance, calcium-activated potassium channel HsapBK, was studied (paper I and II). In paper I, a high-resolution solution NMR structure of HsapBKp in detergent micelles is presented revealing a helix-turn-helix motif. Small structural differences between HsapBKp and KvAPp, positioning the arginines differently, are presented. These structural differences may explain why BK channels are weakly voltage-gated. In paper II, it is shown that HsapBKp perturbs the membrane more than KvAPp and that the membrane perturbation is related to β-structure and to dynamics in the turn in the helix-turn-helix motif.

Second, the membrane interaction of HAMP domains modulating transmission in prokaryotic transmembrane signaling was studied (paper III). Based on the membrane interaction of the AS1 segments of the HAMP domains, two groups were identified: one strongly membrane interacting and one weakly membrane interacting. The two groups are suggested to use different signaling mechanisms.

Third, nonspecific binding of proinsulin C-peptide, the linker peptide connecting chain A and B in insulin, to model membranes was studied (paper IV). The study revealed that C-peptide binds to a model membrane at low pH, but the membrane induces no large structural rearrangements of the peptide. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2012. , 59 p.
Keyword [en]
peptide-lipid interaction, paddle domain, voltage gating, voltage sensor domain, micelle, phospholipid bicelle, solution structure, HAMP domain, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, nonspecific interaction
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-79051ISBN: 978-91-7447-564-7 (print)OAI: oai:DiVA.org:su-79051DiVA: diva2:546714
Public defence
2012-09-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2012-09-06 Created: 2012-08-24 Last updated: 2012-08-29Bibliographically approved
List of papers
1. 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
2. Membrane-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K+ channels
Open this publication in new window or tab >>Membrane-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K+ channels
2012 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 19, 3982-3992 p.Article in journal (Refereed) Published
Abstract [en]

Voltage-gated K+ channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a “paddle” domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the Kv channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic–hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.

National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-79049 (URN)10.1021/bi300188t (DOI)000303961400005 ()
Funder
Swedish Research Council, 621-2011-5964
Available from: 2012-08-24 Created: 2012-08-24 Last updated: 2017-12-07Bibliographically approved
3. Structural characterization of AS1-membrane interactions from a subset of HAMP domains
Open this publication in new window or tab >>Structural characterization of AS1-membrane interactions from a subset of HAMP domains
2011 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1808, no 10, 2403-2412 p.Article in journal (Refereed) Published
Abstract [en]

HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZ(Ec), the E. coli nitrate/nitrite sensor NarX(Ec), and the aspartate chemoreceptor of E. coli (Tar(Ec)). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in alpha-helicity within AS1 from NarX(Ec) and Tar(Ec) but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from Tar(Ec) associated with acidic bicelles. The bulk of AS1 formed an amphipathic alpha-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1-membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors.

Keyword
HAMP domain, Signal transduction, Transmembrane communication, AS1-membrane interactions, Helix-interaction model
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-66515 (URN)10.1016/j.bbamem.2011.06.018 (DOI)000294982000008 ()
Funder
Swedish Research Council, 621-2011-5964
Note

authorCount :3

Available from: 2011-12-27 Created: 2011-12-20 Last updated: 2017-12-08Bibliographically approved
4. pH-Dependent Interaction between C-Peptide and Phospholipid Bicelles
Open this publication in new window or tab >>pH-Dependent Interaction between C-Peptide and Phospholipid Bicelles
2012 (English)In: Journal of Biophysics, ISSN 1687-8019, 185907- p.Article in journal (Refereed) Published
Abstract [en]

C-peptide is the connecting peptide between the A and B chains of insulin in proinsulin. In this paper, we investigate the interaction between C-peptide and phospholipid bicelles, by circular dichroism and nuclear magnetic resonance spectroscopy, and in particular the pH dependence of this interaction. The results demonstrate that C-peptide is largely unstructured independent of pH, but that a weak structural induction towards a short stretch of β-sheet is induced at low pH, corresponding to the isoelectric point of the peptide. Furthermore, it is demonstrated that C-peptide associates with neutral phospholipid bicelles as well as acidic phospholipid bicelles at this low pH. C-peptide does not undergo a large structural rearrangement as a consequence of lipid interaction, which indicates that the folding and binding are uncoupled. In vivo, local variations in environment, including pH, may cause C-peptide to associate with lipids, which may affect the aggregation state of the peptide.

National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-79050 (URN)10.1155/2012/185907 (DOI)
Funder
Swedish Research Council, 621-2011-5964
Available from: 2012-08-24 Created: 2012-08-24 Last updated: 2013-04-29Bibliographically approved

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