The structure of human procathepsin S [Elektronische Ressource] : crystallographic investigations on the functional role of the propeptide / von Guido Kaulmann

Guido Kaulmann

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2004
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

The Structure of Human Procathepsin S:
Crystallographic investigations on the functional role
of the pr opeptide
Dissertation
zur Erlangung des akademischen Grades
Doc tor rerum na turalium ( Dr . rer. nat.)
V orgelegt dem
R at der Biologi sch- Phar mazeut ischen Fakul tät
der Friedrich- Schiller-U ni ver sität Jena
von D ipl. B iol. Gui do K aul mann
gebor en am 20.01.1966
in Bielef eldGutachter :
1. Prof. Dr. Bernd Wi ederanders
2. Prof. Dr. Rolf Hilgenfeld
3. Prof. Dr. D ieter Brőmme--------------------------------------------------------C ontents
Contents Page
Zusammenfassung 1

1. Introduction 4
1.1 Cathepsin S : tissue distribution and function 4
1.2 Synthesis, transport an d activation 5
1.3 Structure of cathepsin S 6
1.4 The propeptide of cathepsin S 6
1.4.1 Primary s truc ture and hom ology 6
1.4.2 The prope ptide as inhi bitor 8
1.4.3 Folding c ompetenc e 8
1.4.4 The ER(F/W )N(I/ V )N-m otif and t he hydrophobi c core 9
1.5 Aim of th e thesis 10
2. Materials 11
2.1 Molecular biology 11
2.1.1 V ectors and pl asmids 11
2.1.2 Bacterial stra ins, m edia 11
2.1.3 Kits 11
2.1.4 Enzymes and buffers 12
2.1.5 Oligonuc leotide s 12
2.2 Bac terial expression 13
2.2.1 Stra in a nd induc er 13
2.2.2 Buffers for c ell lysis 13
2.3 Bac ulovirus expression 13
2.3.1 Ins ect cell culture 13
TM
2.3.2 Bac-N-Blue Kit (Invi troge n) 13
2.3.3 M edia and s upplements 13
2.3.4 Cul ture equi pment 14
2.4 SDS-Page and We stern blot 14
2.4.1 Antibodi es 14
2.4.2 Buffers and s olutions 14
2.4.3 Protein puri fication 14
2.5 Materials for crystallization and cryotools 15
2.6 Additional equipment 15
2.7 Li st of suppliers in alphabetical order 16
i--------------------------------------------------------C ontents
3. Methods 18
3.1 Cloning 18
3.1.1 Cl oni ng of t he hum an proc athepsin S ge ne 18
3.1.1.1 Plasmid prepa ra tion 18
3.1.1.2Amplification of t arget DNA us ing pol ymera se cha in reaction (P CR) 18
3.1.1.3 Site-directed m utage nesis 20
3.1.1.4 Selection of P CR produc ts for l igation a nd t ra nsform ation 21
3.1.1.5D esalting of pl asmid DNA 21
3.1.1.6Ligation of P CR produc ts w ith t he baculovi rus tra nsfe r vector 21
3.1.1.7Tra nsform ation of c ompetent E. coli cells 22
3.1.2 Cl oni ng of t he cathepsin S prope ptide 22
3.1.3 Seque nc ing 22
3.2 Exp ression 23
3.2.1 Expre ssion of re combina nt hum an proc athepsin S in insect cells 23
3.2.1.1Cell culture 23
3.2.1.2Tra nsfe ction of i nsect cells and hom ologous recombina tion betwen t he 23
tra nsfe r vector a nd l inearized Bac-N-Blue AcM NPV DNA
3.2.1.3 Isolation of re combina nt virus (Plaque assay) 24
3.2.1.4PCR a na lysis of recombina nt vira l DNA 25
3.2.1.5Prepa ra tion of hi gh-t iter vi ra l stoc ks 28
TM
3.2.1.6Infection of H ighFive insect cells and prot ein expre ssion of re combina nt 28
protein
3.2.1.7 29Large-scale expre ssion of t he hum an proc athepsin S Cys 25 Ala m utant
3.2.2 Expre ssion of t he cathepsin S prope ptide in E.coli 29
3.3 Purification 30
3.3.1 Puri fication of t he hum an proc athepsin S (Cys 25 Ala) m utant 30
3.3.2 Puri fication of t he hum an c athepsin S prope ptide 30
3.3.3Protein a na lysis 31
3.4 Crystallization 32
3.5 Collection of diffraction data 32
3.5.1 D ata collection of t he cathepsin S prope ptide crystals 32
3.5.2 D ata collection of proc athepsin S crystals 32
3.6 Data p rocessing 33
3.7 Structure solution and refinement 33
3.7.1 33Proc athepsin S Cys 25 Ala m utant
3.7.2 Ca thepsin S prope ptide 33
3.8 Chemical geometry an d interactions 34
3.9 Electrostatic potential 34
ii
fififi--------------------------------------------------------C ontents
4. Results 35
4.1 From th e gene to the purified recombinant protein 35
4.1.1 35Expre ssion of hum an proc athepsin S and its Cys 25 Ala/Ser m utants
4.1.1.1Some de tails on t he baculovi rus expre ssion s ystem 35
4.1.1.2Cl oni ng s tra tegy, pre pa ra tion a nd a na lysis of recombina nt virus 36
4.1.1.3 38Large-scale expre ssion of t he hum an proc athepsin S Cys 25 Ala m utant
4.1.2 40Puri fication of t he hum an proc athepsin S Cys 25 Ala m utant
4.1.3 Bacterial expre ssion a nd puri fication of t he hum an c athepsin S prope ptide 40
4.2 Crystallization and structure determination 41
4.2.1 The crystallization of t he cathepsin S prope ptide 41
4.2.1.1Stra tegy 41
4.2.1.2 Crys tallization us ing t he hangi ng-drop va por di ffus ion m ethod 41
4.2.1.3Crys tallization of t he cathepsin S prope ptide us ing t he counter diffus ion 46
method
4.2.1.4Col lection of di ffraction da ta and proc essing 49
4.2.1.5Attempts to determine the struc ture of the cathepsin S prope ptide 50
4.2.2 51Struc ture de termina tion of t he hum an proc athepsin S Cys 25 Ala m utant
4.2.2.1 Crys tallization 51
4.2.2.2 Col lection of di ffraction da ta and proc essing 51
4.2.2.3Struc ture de termina tion a nd refinement 53
4.2.2.4Qua lity of t he struc ture m odel 54
4.2.3 57The struc ture of the hum an proc athepsin S Cys 25 Ala m utant
4.2.3.1Overa ll de scription 57
4.2.3.2The enzyme dom ains of proc athepsin S 59
4.2.3.3 Overa ll de scription of t he prope ptide 61
4.2.3.4The globul ar dom ain of t he prope ptide 62
4.2.3.5Stabilization of t he globul ar prope ptide dom ain 65
4.2.3.6Anc hori ng of t he prope ptide ont o t he enzyme dom ains 67
4.2.3.7 70Int era ctions be twen helix 3p and t he S ´- s ubs ites
4.2.3.8Int era ctions w ithin t he S subs ites 70
4.2.3.9 Electrostatic pot ential 72
iii
fifiafififi--------------------------------------------------------C ontents
5. Discussion 77
5.1 Crystallization as implication for an independently fol ded structure of the 77
cathepsin S propeptide
5.1.1 A nove l crystallization a pproa ch 77
5.1.2 Ostwald ripeni ng 79
5.1.3 Count er diffus ion t echni que 80
5.1.4 How is the isolated cathepsin S prope ptide struc ture d ? 81
5.2 Procathepsin S structure and the functional role of the propeptide 81
5.2.1 Ca thepsin S-specific intera ctions be twen t he prope ptide and t he enzyme 81
5.2.2 Inhi bition by t he prope ptide and s electivi ty 82
5.2.2.1 The func tiona l rol e of Arg141 and P he146 in c athepsin S 83
5.2.2.2 Inhi bitory a ctivi ty by t he C-termina l part of the prope ptide 84
5.2.3 Cons eque nc es for t he cathepsin S prope ptide struc ture after m utation of 88
conserve d residues of t he ER(F/W )N(I/ V )N-m otif
5.2.4 Electrostatic pot entials and t heir infl uenc e on t he activi ty of c athepsins 90
6. References 92
iv-------------------------------------------------------Table I ndex
Table Index Page
Table 4-1Crys tal grow th s tatistics 43
Table 4-2D ata collection s tatistics for t he cathepsin S prope ptide 49
Table 4-3M atthews coefficients for t he cathepsin S prope ptide 50
Table 4-4D ata collection s tatistics for t he hum an proc athepsin S 52
Cys 25 Ala m utant
Table 4-5M olecular replacement statistics for t he hum an proc athepsin S 53
Cys 25 Ala m utant
Table 4-6 54Refinement statistics for t he hum an proc athepsin S Cys 25 Ala m utant
Table 4-7Stabilizing s alt bridges and hydroge n bonds w ithin t he prope ptide 65
Table 4-8Isoelectric poi nts of cathepsins 73
Table 5-1Prope ptide residues of the C-termina l pa rt 88
v
fififi------------------------------------------------------F igure I ndex
Figure Index Page
Figure 1-1 Struc ture s of proc athepsins L and K 7
Figure 1-2Seque nc e alignm ent of prope ptide s 9
Figure 3-1 34D efini tion of C-H ··· -intera ctions
Figure 4-1Cl oni ng s tra tegy for t he ge ne enc odi ng t he hum an proc athepsin S Cys 25 36
Ala m utant
Figure 4-2Recombina tion s tra tegy 37
Figure 4-3 PCR a na lysis of DNA from recombina nt vira l clone s 37
Figure 4-4W estern bl ot ana lysis of e xpre ssed hum an proc athepsin S 38
Figure 4-5SD S-PAG E ana lysis of the puri fied hum an proc athepsin S 39
Cys 25 Ala m utant
Figure 4-6Restriction a na lysis of pA SK75-hppCS 40
Figure 4-7SD S-PAG E ana lysis of puri fied prot ein s amples under dena turi ng 40
condi tions
Figure 4-8Crys tallization s tra tegy us ing t he vapor di ffus ion m ethod (ha ngi ng drop) 42
Figure 4-9D rops w ith c athepsin S prope ptide crystals in diffe rent num bers 44
Figure 4-10 Ca thepsin S prope ptide crystals 45
Figure 4-11 Count er diffus ion t echni que us ing t he G ra na da Crys tallization Box (G CB)