Analysis of coiled coil oligomerization [Elektronische Ressource] : a multidisciplinary approach / by Carsten C. Mahrenholz

Description

AnAlysis of Coiled Coil oligomerizAtion -A multidisCiplinAry ApproACh Inaugural-Dissertationto obtain the academic degreeDoctor rerum naturalium (Dr. rer. nat.)submitted to the Department of Biology, Chemistry and Pharmacyof Freie Universität Berlin by Dipl. Biol. CARSTEN C. MAHRENHOLZ, MBAborn in Wiesbaden Berlin, September 2010Research for this thesis was conducted from 2007 to 2010, supervised by Dr. Rudolf Volkmerat the Molecular Libraries Group, Charité Berlin.st1 Reviewer: Prof. Dr. Hans-Dieter Volk, Institute of Medical Immunology, Charité Berlinnd2 Reviewer: Prof. Dr. Beate Koksch, Institute for Chemistry and Biochemistry, Freie Universität Berlin date of defence ____________This work was generously funded by the Manchot Foundation (Henkel KGaA) and sup-ported by scholar- and fellowships from the Federation of the Societies of Biochemistry and Molecular Biology, the Charité Medical School, and GlaxoSmithKline.molecular librariesand recognition groupThis work would not have been possible without the kind support of: Rudolf Volkmer, Christiane Landgraf, Ines Kretzschmar, Victor Tapia, and all group members of the Molecular Libraries, Charité, Berlin; Ulrich Bodenhofer, Sepp Hochreiter, Johannes Kepler University, Linz.

Sujets

• Naturwissenschaften

Informations

• Publié par: freie_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 37
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait

A nAlysis   of C oiled C oil o ligomerizAtion - A m ultidisCiplinAry A pproACh

 

Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.)

submitted to the Department of Biology, Chemistry and Pharmacy of Freie niersität Berlin

 

by  

Dipl. Biol. C C.  , B

born in iesbaden

 

Berlin, eptember 

 st eieer

 nd eieer  

esearch for this thesis as conducted from  to , superised by Dr. udolf olmer at theMolecular Libraries Group, Charité Berlin.

Prof. Dr. ans-Dieter ol,Institute of Medical Immunology,  Charité Berlin

Prof. Dr. Beate osch,Institute for Chemistry and Biochemistry,  Freie Uniersität Berlin 

         

date of defence 

 

molecular libraries and recognition group

is or ould not hae been possible ithout the ind support

of udolf olmer, Christiane andgraf, Ines retschmar,

ictor apia, and all group members of theMolecular Libraries,

Charité, Berlin lrich Bodenhofer, epp ochreiter,ohannes epler Uniersity, Lin. Beate osch,Freie Uniersität, Berlin  andro eller,Leibnit Institute for Molecular harmacology, Berlin ichel teinmet,aul cherrer Institute, illigen olger trauss,

oo ordis, openhagenand iolaus rnsting,umboldt Uni

ersity, Berlin. ictor reiff, ohannes cstein, ichal ruil,

Prisca Boisguérin, and icole ittenbring contributed aluable

comments on the manuscript.

y special thans go to Ingrid bfalter for moral support and many

fruitful and stimulating discussions that influenced this or.

is or as generously funded by the

Manchot Foundation enel Gaand sup-

ported by scholar- and felloships from the

Federation of the ocieties of Biochemistry and

Molecular Biology, theCharité Medical chool,

andGlaomithline .

  

  relatioding thednretsnaenu  a ceeiotsen eetrp nihsneb p reathe gof tone i  suterrtcudns  in e nci. gyloiob ni segnellahc  he tas cofe he tibu tiu suolioced coil motif, srtcuutera dn  occurrence hae been described in etensie detail, it might stand to reason that e hae a clearly dran picture of coiled coils. oeer, the rules for oligomeric formation, and thus the ey to biological function, are poorly understood.

is or inestigates the oligomeriation of coiled coils by means of a multidisciplinary approach that combines biochemistry, biophysics, and bioinformatics to shed ne light on the formation of to- and three-stranded coiled coils

Based on comprehensie peptide libraries of C and other coiled coil mutants, the influence of amino acid substitutions on their associa - tion is eamined. Furthermore, this or uses a machine learning ap - proach to tacle coiled coil oligomeriation and identify its underlying rules in the form of eighted amino acid patterns. ese rules form the basis of the highly reliable classification tool PrCoil, hich also isualies the contribution of each indiidual amino acid to the oerall oligomeric tendency of a gien coiled coil seuence.

us, for the first time, a complete netor of seuence parameters that influence oligomeriation is established, and the underlying rules of coiled-coil formation are presented.

is or is rounded off by a methodical contribution. In order for a method to proide a basis for draing sound conclusions, it must be reieed carefully. In the case of peptide libraries, little is non about the cross-reactiity beteen peptides and detection agents.  systematic reie and appraisal of the potential of three common read-out systems – ()-, FIC, and biotinstreptaidin-PD – to cross-react ith indiidual amino acids in a peptide seuence is therefore presented.

I

  

D asänster rut tu - end un ieto neno rrP n  Beiehudnis dereh neugn ics iguther de n geun  mI .eigoloiB ne  gro dereineist drresuofeareß n Falle des eit erbreiteten Coiled- Coil-otis sind speiell trutur und orommen detailliert beschrie - ben. s ist also naheliegend, on einer ollständig aufgelärten trutur ausugehen. m so erstaunlicher ist aber, dass die Coiled-Coil-ligo - merisierung – entrales riterium für die biologische Funtion dieser Proteine – naheu unerstanden ist.

In dieser rbeit ird das Phänomen der Coiled-Coil-ligomerisierung anhand eines multidisiplinären nsates untersucht. rst die ombi - nation aus Biochemie, Biophysi und Bioinformati erlaubt es, die For - mation on ei- und dreisträngigen Coiled-Coils u erlären

u diesem ec ird auf Basis on umfangreichen Peptidbibliothe - en on C und anderen Coiled-Coil-utanten der influss on minosäure-ubstitutionen auf das ssoiationserhalten untersucht. eiterhin beschäftigt sich die orliegende rbeit mit der ntersuchung des ligomerisierungserhaltens on Coiled-Coils. Basierend auf einer neuen eorie und unter uhilfenahme on upport ector aschi - nen erden die der ligomerisierung ugrundeliegenden egeln prä - sentiert. Diese egeln, in Form on geichteten Beiehungen ischen minosäuren, bilden die rundlage eines neuartigen lassifiations- ools. PrCoil ist in der age, die töchiometrie on Coiled-Coils mit außergeöhnlicher enauigeit orherusagen und den Beitrag einelner minosäuren dau u isualisieren. In Form eines eters on euenparametern ird hier erstmalig ein odell eingeführt, das in der age ist, die Coiled-Coil ligomerisierung u erlären. us methodischer icht feit die nendung einer tandard-ethode nicht or ritischer efleion. nabdingbar für eine uerlässige Inter - pretation on Peptidbibliotheen ist das issen um potenielle reu - reatiität on membrangebundenen Peptiden mit den acheisrea - genien des nalyten. Daher beinhaltet diese rbeit als dritten Focus eine Begutachtung und Beertung on drei in diesem usammenhang häufig genutten acheissystemen. ()-, FIC und Biotin treptaidin-PD erden auf ihre reureatiität mit einelnen mi - nosäuren in Peptidseuenen hin untersucht.

II

           

A bbreviAtions  

A bstrACt   in   germAn  

C ontents  

 

i ntroduCtion  

     

     

           

         

 

 

 

A bstrACt   in   english  

   

 

b oisyhplACi   methods | 3.2  F   C-          

  ..    ..   ..     PDB  ..   B  ..

b emChAliCio   methods | 3.1  P-  ..      ..  P     ..  B      ..        ..

m ethods  

5  

1  

1

b ioinformAtiCs   And   AtstCstiis | 3.3  D     ..  -      ..         ..  

b ioinformAtiCs | 1.2  ---   ..      ..

C oiled   Coils | 1.1     P

   ..    ..

m ioneslest   And o bjeCtives  

spot- synthesis | 1.3   -   ..  I     ..

     

i

iii

ii

C

v

 

   

2   

18     

III

12

8  

1     

1

A nAlysis   of   Coiled   Coil   ioAtizeromigoln | .3 3  D   ..   P      ..         ..   P   ..      ..     C   PC  .. 

I

1

5

8

   

           

A ppendix  

 

 

 

 

 

 

C urriCulum   vitAe  

l ist   of   figures   And   tAbles  

p oisnulbCitA  

5

     

             

     

                   

2         

2

           

    ..      ...  C    ...     ...  P   ...   P  ...

C ross - reACtivity   of   deteCtion   systems | .1  B  -PD  ..     ..  FIC  ..  ()-  ..  I   ..  P-   ...  C   ...  D     ...  C   -  ...

r esults   And d sissuCnio  

38  

A nAlysis   of   Coiled   Coil   CossAnoitAi | .2  -   ..  D-   ..

b iblAphyiogr  

C losing   remArks  

C nCosonsilu  

xii

x



viii

B  B CCD I FD h I PC IB DI-F  PDB PD P P I P  B t   

Boc DIC DF D  FIC Fmoc Bt P Pfp tBu Pbf PyBP   B tBu F rt

la rg sn sp Cys ln lu ly is Ile

   D C     I



basic local alignment search tool blocs substitution matri camera charging deice enyme-lined immunosorbent assay false discoery rate hydrophobic human immunodeficiency irus high-performance liuid chromatography library for support ector machines matri-assisted laser desorption ioniation - time of flight molecular eight protein data ban peroidase partial specific olume potential support ector machine signal intensity surface plasmon resonance support ector machine tris-buffered saline ildtype

C 

tert-butyloycarbonyl diisopropylcarbodiimide dimethylformamide dimethylsulfoide fluorescein-substituted thiourea deriatie (-fluorenyl)methoycarbony -hydroybenotriaole -methyl--pyrrolidone pentafluorophenyl ester tert-butyl ester , , , , -pentamethyldihydrobenofuran--sulfonyl benotriaolyloy-tris(pyrrolidino)phosphonium heafluorophosphate -(and )-carboytetramethylrhodamine -(ly-),,aio-l--benotrulfartetmuinorulhyetamtrte-,eoratorob tert-butyl trifluoro-acetic acid trityl

alanine arginine asparagine aspartic acid cysteine glutamine glutamic acid glycine histidine isoleucine

eu ys et Phe Pro er r rp yr al

 

   F P     

leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine aline



i uCodontinrt

C   . n  I ce t ino   i . l a . P,  cc g e   m  o P s u a l b i   nn t g i   h  a e u h l   i n  firCric, bidet htsd serc  e e  a e t     rp s a t l o r . t    h t  e f      t n   b d o o r  a etuuc  , tto F p   yci   l e a aed  fo e o eampl l r i C o   c .     l c a i c   il . eh- C . a structural motif, being commonly described as consisting of beteen to and seenaof the proteins in the Protein-helices. lmost  Data Ban (PDB) Bernsteinet al.,  contain coiled coil regions adleyet al.of hich more than  sho dimeric or tri -, , meric interactions. Due to their ability to oligomerie, coiled coils per - form, either on their on or as part of larger protein complees, a ariety of important cellular functions Burhardet al., . eir ubi- uity and the stable interactions of their helices mae coiled coils ideal building blocs for designing noel proteins. Furthermore, coiled coil interactions hae recently attracted attention as promising drug targets trausset al.successful inhibition of membrane fu -, . eir use in sion proteins of iruses such as I Bianchiet al.,  and aian in- fluena ussellet al.supports the concept of rational drug design,  based on coiled coil proteins cFarlaneet al., . oadays, coiled coils are used etensiely and successfully to rationally design multi- stranded structures for applications including basic research, biotechnol - ogy, nanotechnology, material science, and medicine. e ide range of applications and the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomeriation.  page 



Figure 1 | Schematic representation and struc - ture of the parallel dimeric coiled-coil mo - tif. (A) elical heel diagram looing don the helical ais from the - to the C-terminus. eptad positions are labeleda–g anda–g res- pectiely. Positionsa,d,e, andgare color-coded. (B)In the side ie, the helical bacbones are represented by cylinders, the side chains by n - obs, and the path of the polypeptide chain is indicated by a line rapped around the cylin - ders. For simplicity, the supercoiling of the he - lices is not shon. hile residues at positionsa  (purple) andd(blue) mae up the hydrophobic interface, residues at positionse (orange) andg  (red) pac against the hydrophobic core. ey can participate in interhelical electrostatic in - teractions beteen residue i (g of one position) heli and residue i of the other heli (e po- sition in the net heptad), as indicated by the hatched bars. lso indicated is the corea posi- tion (green), hich is often occupied by polar residues mediating strand-pairing specificity. (Figure adapted from asonet al.)

    ..

oday, a plethora of information about coiled coils is aailable, including their prealence, seuence characteristics, and struc - tures. s illustrated inFigure 1, they hae in common a peri - odically recurrent seuence called a heptad repeat of the form (abcdefg) n . sually, the positionsaanddin these repeats are occupied by hydrophobic amino acids located at the hydrophobic core crucial for tertiary structure, hile positionseandgtypically are charged residues ’heaet al., .

     A

 B

ese obious regularities are used to predict coiled coil segments in amino acid seuences upaset al.,  Deloreniet al.,  c- Donnellet al., . ence, one might epect our understanding of coiled coils to be complete. ost remarably, hoeer, the hidden and more comple rules for oligomeric formation, and thus the ey to bi - ological function, are poorly understood.  first but crude indicator of hether the oligomeric state of a coiled coil is dimeric (Figure 2) or trimeric (Figure 3) may be its intra etra-cellular prealence and - upaset al., , but it clearly does not proide any information about the seuence features that goern oligomeriation.

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

Figure 2 | Eamples and helical heel diagram of dimeric coiled coils. are both the Displayed complete proteins and details of the a-helices of (A) and tropomyosin() the D-bound c- unc-un P dimer. ropomyosin mediates the interactions beteen the troponin comple and actin so as to regulate muscle contraction eis et al., . e c-un oncoprotein is a maor component of the transcription factor comple P-, hich regulates the epression of multiple genes essential for cell proliferation, differentiati - on, and apoptosis artlet al., .(B)elical heel diagram of a dimer looing don the he - lical ais from the - to the C-terminus. eptad positions are labeled from a to g. rros repre - sent the interhelical electrostatic interactions.

Despite etensie eperimental and computational efforts such as mutation analysis, , -ray crystallography, and statistics upaset al.,  Portichet al.,  ingraset al.,  heriffet al.,  uet al., , our noledge of hich oligomer a specific coiled coil forms has, until no, been limited to describing the phenomenon on the basis of a small number of protein samples.

      B A

     B A 

Figure 3 | Eamples and helical heel diagram of trimeric coiled coils.Displayed are both the complete proteins and details of thea-helices of(A) the surface transmembrane glycoprotein (P) of the ebola irus and()mannose-bin- ding lectin (B). P is responsible for bin - ding to target cells and subseuent fusion of the iral and host-cell membranes alasheichet al., . B is a calcium-dependent serum protein that plays a role in the innate immune response by binding to carbohydrates on the sur - face of a ide range of pathogens urner, . ll figures ere created using Py (httppy - mol.sourceforge.net) (B)elical heel diagram of a trimer looing don the helical ais from the - to the C-terminus. eptad positions are labe - led from a to g. rros represent the interhelical electrostatic interactions.

o, as the amount of aailable post-genomic seuence data is groing rapidly, the challenge is to eplain coiled coil oligomeriation by etract - ing an actual set of rules from this data.

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