incorporating nonproteinogenic amino acids into azurin and - Ideals

217 Pages·2010·10.39 MB·English

by  Kevin Clark

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ABSTRACT
Metalloproteins play important roles in biological systems since metal-binding sites are
found in ~ 1/3 of structurally characterized proteins and in ~ 1/2 of all proteins. Despite progress,
the role of many metal-binding sites remains poorly understood. Furthermore, their detailed
geometric and electronic structure has yet to be fully elucidated. Site-directed mutagenesis using
natural amino acids has continued to be the primary method of probing of metal-binding sites.
However, it has become increasingly evident that site-directed mutagenesis, though powerful,
cannot address the precise role of the metal ligands without changing multiple factors because of
limited number of natural amino acids that allows isostructural substitutions. The relative paucity
of functional group availability within the proteinogenic amino acids also limits ability to fine
tune and expand protein’s activity. To address the need to precisely determine specific metal
binding ligand functionalities, and further efforts to engineer new function into proteins,
expressed protein ligation (EPL) has been used to introduce non-proteinogenic amino acids to
metalloproteins, such as the Type 1 (T1) copper site of azurin and the Cu site found in
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engineered Cu Azurin to aid the understanding of the roles of each ligand in this electron
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transfer protein.
This thesis covers the replacement of two copper-binding ligands in azurin, Cys112 and
Met121, with nonproteinogenic amino acid analogues. Investigating the Cys112(S)-Cu Ligand-
to-Metal Charge Transfer (LMCT) that gives azurin its intense blue color and its signature
absorbance at 625 nm in the visible spectrum using two different analogues has been performed.
Incorporating selenocysteine for Cys112 resulted in a new variant that maintained type 1
geometry , making it possible to elucidate the electron density of the site with more precision in
the future. To further expand this concept, EPL was used to incorporate homocysteine (Hcy) to
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investigate the rack hypothesis for the type 1 copper center. Insertion of a Cys derivative with an
additional methylene group demonstrated that the type 1 site in azurin was capable of adjusting
sufficiently through protein folding to maintain type 1 geometry and function. Together, these
mutations provide insight into the unique relationship between the site’s geometric features and
its electronic features by minimally perturbing the site and selectively altering each feature
individually.
Secondly, while conversion of blue copper sites with a weak axial ligand to green copper
sites containing a medium strength axial ligand has been demonstrated in cupredoxins,
converting blue copper sites to a red copper site with a strong axial ligand has not been reported.
By replacing the weak Met121 thioether from azurin with a strong thiolate ligand from Hcy, a
variant was created which had spectra (R = 1.5, and A = 180 x 10-4 cm-1) and a Cu-S(Cys)
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distance (2.22 Å) very similar to that of the red copper protein nitrosocyanin. The results
strongly support the “coupled distortion” model, which helps explain axial ligand tuning of
spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino
acids to address critical chemical biological questions.
Lastly, the unique Cu coordination of a backbone carbonyl in Cu azurin site was
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investigated using backbone carbonyl analogues of Glu. By replacing the carbonyl of the natural
peptide linkage with an ester moiety, we have shown that the basicity of the coordinating
carbonyl seems not to play a major role in either the spectroscopic or reduction potential features
of the Cu site. These studies have provided a unique methodology for investigating metal ligand
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interactions beyond the standard side chain interactions.
In summary, a means to investigate the specific function of individual metal ligand
residues in azurin and Cu azurin by using EPL to incorporate nonproteinogenic amino acid
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analogues has been demonstrated. This work has resulted in new insights into the role of the
metal ligands and has shaped future metalloprotein engineering efforts.
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To my wife Eve whose support and love got me through this research.
I love you.
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ACKNOWLEDGEMENTS

First I wish to thank my advisors, Wilfred van der Donk and Yi Lu. Thank you Wilfred
for being a phenomenal advisor, mentor, and friend. Without your guidance, patience and
willingness to help, I could not have completed the work described in this thesis. I will always be
grateful for our conversations both about science and the nuances of life outside the lab that
helped shape my journey through graduate school. Thank you Yi for being an exceptional
mentor. Thank you for all of your guidance, understanding and teaching me how to be
professional. Also, thanks to my committee members, Eric Jakobsson and Antony Crofts, who
provided much support and guidance.
Thank you to all of the past and current members of the Lu group and van der Donk
group whom I have had the honor working with for insightful conversations and helpful
discussions. Thank you to Cyril Jacquot for being a wealth of knowledge and an immutable
friend. Also, thanks to Noah Bindman, Patrick Knerr, and Lindsey Johnstone whose interesting
chats always led to fun times in the lab. I will miss all the laughs and singing while in the lab.
Thank you also to Lu group members Natasha Yeung, Yang Yu, Nicholas Marshall, Nathan
Sieracki, and Kyle Miner for great discussions.
Thank you Martha Freeland, for being a lifeline during my tenure at UIUC. Thanks for
being there for everything from supporting lab experiments to emotional support and medical
aid. You will remain a great friend forever.
Thanks to my wife, Eve, and my dog, Einstein, who endured this long process with me,
always providing laughs and love. Lastly, thanks to my family, especially my mother, Mary, and
my extended family, my father-in-law, Albert, and mother-in-law, Tessie for all their support.
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TABLE OF CONTENTS

LIST OF ABBREVIATIONS..................................................................................................................ix

LIST OF FIGURES.................................................................................................................................xii

LIST OF SCHEMES...............................................................................................................................xiv

LIST OF TABLES...................................................................................................................................xv

CHAPTER 1: INTRODUCTION............................................................................................................1
1.1 Transition metals in biology.................................................................................................1
1.2 Copper proteins.....................................................................................................................3
1.3 Type I copper proteins..........................................................................................................5
1.4 Azurin from Pseudomonas aeruginosa................................................................................7
1.5 Cu azurin.............................................................................................................................11
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1.6 Methods of nonproteinogenic amino acid incorporation......................................................17
1.7 Biosynthetic routes for nonproteinogenic amino acid incorporation....................................18
1.8 Synthetic and semi-synthetic routes for nonproteinogenic amino acid incorporation..........24
1.9 Incorporating nonproteinogenic amino acids into azurin and Cu azurin............................33
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1.10 Summary.............................................................................................................................35
1.11 References...........................................................................................................................36

CHAPTER 2: INVESTIGATING THE RACK HYPOTHESIS: CYS112HCY AZURIN....................50
2.1 Introduction...........................................................................................................................50
2.2 Construction of Cys112Hcy azurin using EPL.....................................................................52
2.3 Spectroscopic characterization of Cys112Hcy azurin..........................................................54
2.4 Electrochemical analysis.......................................................................................................56
2.5 Molecular modeling..............................................................................................................57
2.6 Conclusions...........................................................................................................................59
2.7 Future directions...................................................................................................................60
2.8 Experimental.........................................................................................................................61
2.9 References.............................................................................................................................65

CHAPTER 3: INCORPORATING 77SE-SEC INTO AZURIN: C112U AZURIN................................69
3.1 Introduction...........................................................................................................................69
3.2 Incorporating Sec into azurin: background...........................................................................70
3.3 Optimization of the synthesis of Sec(PMB).........................................................................72
3.4 Solid phase peptide synthesis of C112U azurin 17-mer.......................................................74
3.5 Optimization and semi-synthesis of C112U azurin..............................................................82
3.6 Characterization of selenocysteine azurin............................................................................87
3.7 Synthesis of 77Se-Sec(PMB) and SPPS of 77Se-C112U 17-mer...........................................87
3.8 Semi-synthesis of 77Se-C112U azurin..................................................................................90
3.9 77Se C112U Azurin ENDOR analysis...................................................................................92
3.10 Conclusions.........................................................................................................................94
3.11 Future directions.................................................................................................................94
3.12 Experimental.......................................................................................................................94
3.13 References...........................................................................................................................109

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CHAPTER 4: TRANSFORMING BLUE COPPER TO RED COPPER: MET121HCY AZURIN......114
4.1 Introduction...........................................................................................................................114
4.2 Interconverting Blue and Green Copper Sites by Site Directed Mutagenesis......................116
4.3 Preparation of Met121Cys Azurin........................................................................................117
4.4 UV-Vis Spectral Characterization of Met121Cys Azurin....................................................119
4.5 X-Band EPR Characterization of Met121Cys Azurin..........................................................122
4.6 Preparation of Met121Hcy Azurin.......................................................................................125
4.7 UV-Vis Spectral Characterization of Met121Hcy Azurin....................................................127
4.8 X-Band EPR Characterization of Met121Hcy Azurin.........................................................129
4.9 EXAFS Characterization of Met121Cys and Met121Hcy Azurins......................................131
4.10 Electrochemical Studies......................................................................................................135
4.11 Conclusions.........................................................................................................................138
4.12 Future Directions................................................................................................................138
4.13 Experimental.......................................................................................................................138
4.14 References...........................................................................................................................145

CHAPTER 5: PROBING BACKBONE CARBONYL INTERACTIONS OF CU AZURIN..............153
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5.1 Introduction...........................................................................................................................153
5.2 EPL of WT Cu Azurin........................................................................................................155
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5.3 Synthesis of Backbone Analogue Peptides...........................................................................160
5.4 Installing an Ester Into Cu Azurin......................................................................................161
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5.5 SPPS of Peptide 5.3 By Fmoc Chemistry.............................................................................162
5.6 SPPS of Peptide 5.3 By Boc Chemistry...............................................................................166
5.7 Semi-synthesis of Ester-Cu Az By EPL.............................................................................169
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5.8 Installing a Thioamide Into Cu Azurin...............................................................................174
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5.9 Semi-synthesis of Thioamide-Cu Azurin By EPL..............................................................177
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5.10 Conclusions.........................................................................................................................179
5.11 Future Directions................................................................................................................180
5.12 Experimental.......................................................................................................................181
5.13 References...........................................................................................................................195

AUTHOR’S BIOGRAPHY.....................................................................................................................202
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LIST OF ABBREVIATIONS

ACN...........................acetonitrile
AcOH.........................acetic acid
Asn.............................asparagine
Asp.............................aspartic Acid
ATP............................adenosine triphosphate
Az...............................azurin
Boc.............................tert-butyloxycarbonyl
CBD...........................chitin binding domain
CcO............................Cytochrome c Oxidase
CV..............................cyclic voltammetry
Cys.............................cysteine
DBU...........................1,8-diazabicyclo[5.4.0]undec-7-ene
DCM..........................dichloromethane
DDAB........................dididecldimethylammonium bromide
DFT............................density functional theory
DIC............................N,N'-diisopropylcarbodiimide
DIPEA.......................diisopropylethyl amine
DKP...........................diketopiperazine
DMAP........................4-dimethylaminopyridine
DMF...........................dimethylformamide
DNP...........................dinitrophenyl
DTNP.........................2,2’dithiobis(5-nitropyridine)
DTT...........................dithiothreitol
EDCI..........................1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
EDT...........................ethanedithiol
EDTA.........................ethylenediaminetetraacetic acid
ENDOR.....................electron-nuclear double resonance
EPL............................expressed protein ligation
EPR............................electron paramagnetic resonance
ESEEM......................electron spin echo envelope modulation
ESI-MS......................electrospray ionization mass spectrometry
ET..............................electron transfer
EtOAc........................ethyl acetate
EtOH..........................ethanol
EXAFS.......................extended x-ray absorption fine structure
FL...............................full length
Fmoc..........................fluorenylmethyloxycarbonyl
Gln.............................glutamine
Glu.............................glutamate
Gly.............................glycine
HBTU........................O-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate
HCTU........................O-(1H-6-chlorobenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexafluorophosphate
Hcy.............................homocysteine
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HEPES.......................4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
His..............................histidine
HOBt..........................1-hydroxybenzotriazole hydrate
IMPACT....................intein mediated purification with an affinity chitin-binding tag
LB..............................luria broth
Leu.............................leucine
LMCT........................ligand to metal charge transfer
Lys.............................lysine
MALDI-MS...............matrix-assisted laser desorption ionization mass spectrometry
MeCN........................acetonitrile
MES...........................2-(N-morpholino)ethanesulfonic acid
MESNa......................sodium 2-mercaptoethanethiosulfonate
Met.............................methionine
MOPS........................3-(N-morpholino)propanesulfonic acid
MPAA........................mercaptophenyl acetic acid
mRNA........................messenger RNA
MWCO......................molecular weight cutoff
NCL...........................native chemical ligation
NEB...........................New England Biolabs
NMA..........................N-methylmercaptoacetamide
NMM.........................N-methylmorpholine
NMR..........................nuclear magnetic resonance
NOR...........................nitrous oxide reductase
NpsCl.........................2-nitrophenyl sulfenyl chloride
PAM...........................4-hydroxymethyl-phenylacetamidomethyl
PEG............................polyethylene glycol
PGE............................pyrolytic graphite edge
PMB...........................p-methoxybenzyl
PNBP.........................4-(4-nitrobenzyl) pyridine
PMSF.........................phenylmethanesulfonylfluoride
PS...............................polystyrene
RNA...........................ribonucleic acid
RP-HPLC...................reverse phase high performance liquid chromatography
rR...............................resonance raman
RT..............................room temperature
SDM...........................site directed mutagenesis
SECIS........................selenocysteine insertion sequence
Sec.............................selenocysteine
Ser..............................serine
SPPS..........................solid phase peptide synthesis
TCEP.........................tris-(2-carboxyethyl)phosphine
TFA............................trifluoroacetic acid
TFE............................trifluoroethane
TFMSA......................trifluoromethanesulfonic acid
THF............................tetrahydrofuran
Thr.............................threonine
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