Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2011 The Evolution of Thermal Compensation in Antarctic Fish Parvalbumins Arthur Carl Whittington Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES THE EVOLUTION OF THERMAL COMPENSATION IN ANTARCTIC FISH PARVALBUMINS By ARTHUR CARL WHITTINGTON A dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Summer Semester, 2011 The members of the committee approve the dissertation of Arthur Carl Whittington defended on June 14th, 2011. _______________________________________ W. Ross Ellington Professor Directing Dissertation _______________________________________ Timothy S. Moerland Professor Co-Directi g Dissertatio _______________________________________ Timothy M. Loga U i1ersity Represe tati1e _______________________________________ P. Brya t Chase Committee Member _______________________________________ Ga1i 2.P. Naylor Committee Member Appro1ed, _____________________________________ P. Brya t Chase, Chairperso , Departme t of Biological Scie ce The Graduate School has 1erified a d appro1ed the abo1e- amed committee members. ii ACKNOWLEDGEMENTS I ha1e bee 1ery fortu ate duri g my graduate studies to ha1e such a supporti1e a d 7 owledgeable doctoral committee. I would li7e to tha 7 my co-ma8or professors, Tim Moerla d a d Ross Elli gto . Tim Moerla d accepted me i to his lab, i troduced me to research a d comparati1e biochemistry, a d pro1ided 7ey directio duri g my dissertatio wor7. He has bee a e9celle t teacher, me tor a d frie d. Ross Elli gto too7 me o as his last graduate stude t a d has pro1ided me with sou d guida ce a d has made himself a1ailable at all times despite his busy schedule. The completio a d quality of this dissertatio would ot ha1e bee possible without his participatio . Tim Loga a d Ga1i Naylor ha1e gi1e time a d 1aluable i put throughout my doctoral wor7. I would li7e to tha 7 Brya t Chase for allowi g me to be a foster stude t i his lab group. He has pro1ided ad1ice o research, writi g a d o scie ce i ge eral. Florida State U i1ersity pro1ides a e9celle t le1el of support for research through its core facilities. The A alytical lab, Seque ci g lab, a d Molecular Clo i g facility a d their perso el ha1e pro1ided 1aluable assista ce throughout this pro8ect. All calcium bi di g co sta t measureme ts were made i the Physical Biochemistry Facility of the I stitute of Molecular Biophysics. All stopped-flow measureme ts were performed i the laboratory of Dr. 2o atha Da1is at Ohio State Medical Ce ter. I would also li7e to tha 7 former lab members. Bria Stor: is a great frie d, family ma , a d e9celle t scie tist. Neil Sa scrai te a d 2e ifer Be bow pro1ided frie dship a d assista ce duri g the early stages of my graduate career. Gregg Hoffma has taught me a great deal about protei e9pressio a d purificatio . Theresa Gro1e taught me how to seque ce my first cDNA a d has bee a e9celle t lab mate, collaborator a d frie d. Da ielle Sa do:-Osmus pro1ided assista ce duri g the seque ci g phase of the pro8ect. The late Bruce Sidell made possible the collectio of A tarctic fish specime s that made this pro8ect possible as well as pro1ided 7ey i put duri g the lab’s i itial wor7 o par1albumi . I wa t to gi1e special tha 7s to George Somero, Do al Ma aha a d the NSF I ter atio al A tarctic Biology Trai i g Course for ta7i g me to McMurdo Statio , A tarctica. George is a e9celle t me tor a d pro1ided me with o e of my most cherished lear i g a d life e9perie ces. iii I would li7e to tha 7 my good frie ds i Tallahassee a d 2ac7so 1ille for all their support a d pro1idi g me with a outlet for fu a d rela9atio . I am tha 7ful for the special frie dships I ha1e made with fellow graduate stude ts here at FSU. My sister Christi Whitti gto has bee a collaborator a d o e of my best frie ds. I would li7e to tha 7 Sarah Patto for her lo1e a d frie dship o1er the years. Fi ally, I wa t to tha 7 a d recog i:e my pare ts Cathy a d Curtis Whitti gto for their e1er e di g a d u co ditio al support. i1 TABLE OF CONTENTS List of Tables ................................................................................................................................. 1i List of Figures............................................................................................................................... 1ii Abstract.......................................................................................................................................... i9 1. INTRODUCTION...................................................................................................................1 .. ANCESTRAL SEQUENCE RECONSTRUCTION AND HOMOLOGY MODELING REVEAL POTENTIAL FUNCTIONALLY ADAPTIVE SUBSTITUTIONS IN ANTARCTIC FISH PARVALBUMINS ..............................................................................................................1. I troductio ...........................................................................................................................1. Materials a d methods...........................................................................................................13 Results ...................................................................................................................................17 Discussio ...............................................................................................................................8 3. EXPRESSION, PURIFICATION AND FUNCTIONAL CHARACTERIAATION OF RECOMBINANT PARVALBUMINS..........................................................................................3. I troductio ...........................................................................................................................3. Materials a d methods...........................................................................................................33 Results ...................................................................................................................................3. Discussio ..............................................................................................................................5C 3. CONCLUSIONS...................................................................................................................C0 APPENDIX....................................................................................................................................C3 REFERENCES ..............................................................................................................................77 BIOGRAPHICAL SDETCH .........................................................................................................83 1 LIST OF TABLES Table ..1 Ge Ba 7 accessio umbers for teleost par1albumi cDNA seque ces. .....................15 Table .... Predicted free e ergy of foldi g 1alues for homology models of e9ta t a d reco structed PV seque ces a d predicted effects of 1irtual mutage esis.....................................C Table 3.1. Calcium dissociatio co sta ts for GBPV WT plus muta ts at a ra ge of temperatures...................................................................................................................................51 Table 3... Calcium dissociatio rates measured by stopped-flow spectrometry usi g terbium fluoresce ce as a reporter...............................................................................................................53 Table 3.3. Calcium o -rates for GBPV WT a d muta ts..............................................................55 1i LIST OF FIGURES Figure 1.1 Correspo di g states theory ............................................................................................ Figure 1... E1olutio ary compe satio of protei fu ctio ............................................................3 Figure ..1 A tarctic fish par1albumi protei seque ce alig me t..............................................18 Figure .... A cestral seque ce reco structio guide tree a d method outli e. ............................1E Figure ..3. Reco structed a cestral seque ces shows u ique substitutio patter betwee Perciformes a d otothe ioid a cestral PV seque ces...................................................................1 Figure ..3. Ambiguity i seque ce reco structio . ......................................................................... Figure ..5. Seque ce alig me t of PAPV a d NAPV with the e9ta t GBPV. ..............................3 Figure ..C. Locatio of o -co ser1ati1e substitutio s ide tified by ASR....................................5 Figure ..7. Homology models showi g rei troductio of hydroge bo ds by 1irtual mutage esis.....................................................................................................................................7 Figure ..8. Fu ctio al tu i g by substitutio s away from the acti1e site i GBPV WT ...............E Figure 3.1. Represe tati1e e9ample of fluoresce ce data collected duri g a titratio of 1..5 FM fluo-3 a d 1..5 FM PV ..................................................................................................................38 Figure 3... Represe tati1e fluo-3 calcium Dd determi atio cur1e...............................................30 Figure 3.3. Represe tati1e PV calcium bi di g cur1e ..................................................................31