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Incorporating Nonproteinogenic Amino Acids Into Azurin and Cua Azurin

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Incorporating Nonproteinogenic Amino Acids Into Azurin and Cua Azurin ! ! ! ! ! "#$%&'(!)*+!#$,+-!$.!/+)0,!%&'1&'(!,&(0'1-!&'!23"#+1$4&'-5! 0 &'2$#"$#0)&'(!'$'"#$)+&'$(+'&2!0/&'$!02&1-!&')$!063#&'!0'1!23 !063#&'! ! ! ! ! ! ! %7! ! 8+9&'!/:!2,0#8! ! ! ! ! ! ! ! 1&--+#)0)&$'! ! -;<=>??@A!>B!CDE?>DF!G;FG>FF=@B?!HG!?I@!E@J;>E@=@B?K! GHE!?I@!1HL?HE!HG!"I>FHKHCIM!>B!%>HLI@=>K?EM! >B!?I@!(EDA;D?@!2HFF@N@!HG!?I@! 3B>O@EK>?M!HG!&FF>BH>K!D?!3E<DBDP2ID=CD>NBQ!RSTS! ! ! ! 3E<DBDQ!&FF>BH>K! ! ! ! 1HL?HEDF!2H==>??@@5! ! ! "EHG@KKHE!7>!,;Q!2ID>E!DBA!2HP1>E@L?HE!HG!#@K@DELI! ! ! "EHG@KKHE!U>FGE@A!ODB!A@E!1HBVQ!2HP1>E@L?HE!HG!#@K@DELI! ! "EHG@KKHE!0B?HBM!2EHG?K! ! "EHG@KKHE!+E>V!WDVH<KKHB! 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 CuA site found in engineered CuA Azurin to aid the understanding of the roles of each ligand in this electron 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 ! ""! 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 -4 -1 variant was created which had spectra (RL= 1.5, and A|| = 180 x 10 cm ) and a Cu-S(Cys) 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 CuA azurin site was 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 CuA site. These studies have provided a unique methodology for investigating metal ligand interactions beyond the standard side chain interactions. In summary, a means to investigate the specific function of individual metal ligand residues in azurin and CuA azurin by using EPL to incorporate nonproteinogenic amino acid ! """! 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. ! "#! To my wife Eve whose support and love got me through this research. I love you. ! #! 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. ! #"! 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 CuA azurin.............................................................................................................................11 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 CuA azurin............................33 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
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