Kenneth Mills

mills

Chemistry Department
Anthony and Renee Marlon Professor in the Sciences
Professor of Chemistry
Ph.D., Harvard University

Email: kmills@holycross.edu
Office Phone: 508-793-3380
Office: Haberlin 337
Lab: Smith Laboratories 367
PO Box: C
 

Fields: Biochemistry

 

 

Research Summary

Lab Members

Lab Alumni

Grants

Publications

Lab photos


 

 

Research Summary

Our lab studies protein splicing and the enzymology of extreme thermophiles.

Protein splicing is a post-translational modification. It involves the self-catalyzed excision of an intervening polypeptide sequence, the intein, concomitant to the ligation of the flanking polypeptides, the exteins, via a native peptide bond.

Below is a schematic of the protein splicing process:

We are interested in

a) The means by which non-canonical inteins facilitate the individual steps of protein splicing.

b) The means by which inteins coordinate the steps of splicing.

c) The role that inteins play in the control of extein function.

d) Comparisons of the catalytic strategies of enzymes from thermophiles and other extremophiles and their mesophilic equivalents.

Below is the standard chemical mechanism of protein splicing:

 

 

External Grants
2019-2022      National Institutes of Health, NIGMS, R15: "Conditional Protein Splicing of inteins from extreme thermophiles and halophiles: Structure, Mechanism, and Function."

2015-2019      National Science Foundation, RUI: "RUI: The Catalysis of Protein Splicing and the Structure and Stability of Thermophilic Inteins: An Integrated Research and Education Program"
2015                Research Corporation Cottrell Scholar
2013-2016      National Science Foundation, RUI: "Intein Structure and Catalysis of Protein Splicing and Hedgehog Autoprocessing: An Integrated Research and Education Program"
2010-2013      National Science Foundation, RUI: "Catalysis and coordination of the steps of protein splicing by non-canonical inteins: An integrated research and education program"
2009-2014      Henry Dreyfus Teacher-Scholar Award
2009                ASBMB-UAN Outreach Award
2005-2010      National Science Foundation, CAREER grant, “CAREER: Alternative Mechanisms of HINT domain autoprocessing: An integrated undergraduate research and education program”
2003-2005      National Science Foundation, Major Research Instrumentation, “Acquisition of Protein Purification and Analysis Equipment to Enhance Biochemical Research and Education at the College of the Holy Cross” 
2003-2005      Research Corporation, Cottrell College Science Award, “The Mechanism and Molecular Evolution of Hedgehog Proteins and Related Auto-Processing Domains”
2002-2004      Petroleum Research Fund (American Chemical Society), Type GB, “The Mechanism of Protein Splicing and the Molecular Evolution of Inteins.”
1997-2001      Howard Hughes Medical Institute Predoctoral Fellowship
1995-1996      Barry M. Goldwater Fellowship

(Undergraduate co-authors indicated by asterisk)

39. *Wall, D.A., *Tarrant, S.P., Wang, C., Mills, K.V., and Lennon, C.W. (2021) Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations. Front. Mol. Biosci. 8, 752824, 1-17.
38. *Williams, J.E., *Jaramillo, M.V., Li, Z., Zhao, J., Wang, C., Li, H.,  and Mills, K.V. (2021) An alternative domain-swapped structure of the Pyrococcus horikoshii PolII mini-intein. Scientific Reports, 11, 11680, 1-12.
37. Robinzon, S., *Cawood, A.R., *Ruiz, M.A., Gophna, U., Altman-Price, N., and Mills, K.V. (2020) Protein Splicing Activity of the Haloferax volcanii PolB-c intein is sensitive to homing endonuclease domain mutations. Biochemistry59, 3359-3367.
36. *Chiarolanzio, K.C., *Pusztay, J.M., *Chavez, A., Zhao, J., Xie, J., Wang, C., and Mills, K.V. (2020) Allosteric Influence of Extremophile Hairpin Motif Mutations on the Protein Splicing Activity of a Hyperthermophilic Intein. Biochemistry59, 2459-2467.
35. Zhao, J., Du, Z., Wang, C., and Mills, K.V. (2020) Methods to Study the Structure and Catalytic Activity of cis-splicing Inteins. Methods Mol Biol2133, 55-73.
34. *Ramsoomair, C.K., *Yakely, A.E., *Urbanski, L.M., *Karanja, K., *Giaccone, Z.T., *Siegart, N.M., Wang, C., Gomez, A.V., Reitter, J.N., and Mills, K.V. (2017) Coordination of the third step of protein splicing in two cyanobacterial inteins. FEBS Letters, 591, 2147-2154.
33. Zwarycz, A.S., Fossat, M., Akanyeti, O., Lin, Z., Rosenman, D.J., Garcia, A., Royer, C.A., Mills, K.V., and Wang, C. (2017) V67L Mutation Fills Internal Cavity to Stabilize RecA Mtu Intein. Biochemistry 56, 2715-2722.
32.*Minteer, C.J., *Siegart, N.M., *Colelli, K.C., Liu, X., Linhardt, R.J., Wang, C., Gomez, A.O., Reitter, J.N., and Mills, K.V. (2017) Intein-promoted cyclization of aspartic acid flanking the intein leads to atypical C-terminal cleavage. Biochemistry 56, 1042-1050.
31. Reitter, J.N., *Cousin, C.E., *Nicastri, M.C., *Jaramillo, M.V., and Mills, K.V. (2016) Salt-dependent conditional protein splicing of an intein from Halobacterium salinarumBiochemistry 55, 1279-1282.
30. Mills, K.V. (2015) Biochemistry in an undergraduate writing-intensive first-year program: Seminar courses in drugs and bioethics. Biochemistry and Molecular Biology Education 43, 263-272.
29. Kulevich, S.E., Herrick, R.S., and Mills, K.V. (2014) A Discovery Chemistry Experiment on Buffers. Journal of Chemical Education, J. Chem. Educ. 91, 1207-1211.
28. Mills, K.V., Johnson, M.A., and Perler, F.B. (2014) Protein splicing: How inteins escape from precursor proteins. Journal of Biological Chemistry, 289, 14498-14505.
27. Topilina, N.I. and Mills, K.V. (2014) Recent advances in in vivo applications of intein-mediated protein splicing. Mobile DNA 5, 5.
26. *Nicastri, M.C., *Xega, K., Li, L., Xie, J., Wang, C., Linhardt, R.J., Reitter, J.N., and Mills, K.V. (2013) Internal Disulfide Bond Acts as a Switch for Intein Activity. Biochemistry 52, 5920-5927.
25. Mills, K.V. (2013) Self-Splicing Proteins. In: Handbook of Proteolytic EnzymesThird Edition, Vol. 1., ed. N. Rawlings and G. Salvesen, 315-321.
24. Chen, W., Li, L., Du, Z., Liu, J., Reitter, J.N., Mills, K.V., Linhardt, R.J. and Wang, C. (2012) Intramolecular Disulfide Bond between Catalytic Cysteines in an Intein Precursor. J. Am. Chem. Soc.,134, 2500-2503.
23. Du, Z., Liu, J., Albracht, C.D., Hsu, A., Chen, W., *Marieni, M.D., *Colelli, K.M., *Williams, J.E., Reitter, J.N., Mills, K.V. and Wang, C. (2011) Structural and Mutational Studies of a Hyperthermophilic Intein from DNA Polymerase II of Pyrococcus abyssi. J. Biol. Chem. 286, 38638-38648. (Cover of Nov. 4 2011 issue.)
22. Liu, J., Du, Z., Albracht, C.D., Naidu, R.O., Mills, K.V. and Wang, C. (2011) (1)H, (13)C, and (15)N NMR assignments of the Pyrococcus abyssi DNA Polymerase II intein. BiomolNMR Assign5, 233-235.
21. Reitter, J.N. and Mills, K.V. (2011) Canonical protein splicing of a class one intein that has a class three non-canonical sequence motif. J. Bacteriol. 193, 994-997.
20. *O’Brien, K.M., *Schufreider, A.K., *McGill, M.A., *O’Brien, K.M., Reitter, J.N. and Mills, K.V. (2010) Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein. BiochemBiophys. Res. Commun.403, 457-461.
19. *Kerrigan, A.M, *Powers, T.L, *Dorval, D.M., Reitter, J.N. and Mills, K.V. (2009) Protein Splicing of the Three Pyrococcus abyssi ribonucleotide reductase inteins. Biochem. Biophys. Res. Commun. 387, 153-157.
18. Mills, K.V., Herrick, R.S., Ditzler, M.A., Nestor, L.P., Shafer, H., and Guilmette, L.W. (2008) Introducing Undergraduate Students to Electrochemistry: A Two-Week Discovery Chemistry Experiment. J. ChemEduc.85, 1116-1119.
17.  Herrick, R.S., Mills, K.V., and Nestor, L.P. (2008) Data Pooling in a Chemical Kinetics Experiment: The Aquation of a Series of Cobalt (III) Complexes. J. Chem. Educ. 85, 1120-1122.
16.  Mills, K.V. and Guilmette, L.W. (2007) Thermochemical Analysis of Neutralization Reactions: An Introductory Discovery Experiment. J. Chem. Educ84, 326-328.
15.  Bellin, R.M. and Mills, K.V. (2007) The Holy Cross Biochemistry Concentration: An Integrated Four-Year Program to Develop Undergraduate Research Scholars. In Developing and Sustaining a Research-Supportive Curriculum, ed K.K. Karukstis and T.E. Elgren, Council on Undergraduate Research.
14.  Mills, K.V., Connor, K.R., *Dorval, D.M., and *Lewandowski, K.T. (2006) Protein Purification via Temperature-Dependent, Intein-Mediated Cleavage from an Immobilized Metal Affinity Resin.  Anal. Biochem.356, 86-93.
13.  Mills, K.V., *Dorval, D.M., *Lewandowski, K.T. (2005) Kinetic analysis of the individual steps of protein splicing for the Pyrococcus abyssi PolII intein. J. Biol. Chem 280, 2714-2720.
12. Mills, K.V., *Gareau, J.R., and *Garcia, A.M. (2005) Low-cost modification to the Copacabana method for spreading transformation mixtures.  BioTechniques39188.
11. Mills, K.V., Perler, F.B. (2005) The Mechanism of Intein-Based Protein Splicing: Variations on a Theme.Protein & Peptide Letters, 12, 751-755.
10. Mills, K.V. (2005) Protein Splicing. In Encyclopedia of Molecular Cell Biology and Molecular Medicine,Second Edition, ed, RA Meyers, 241-265, Weinheim: Wiley-VCH. 
9. Mills, K.V., Paulus, H. (2005) Biochemical Mechanisms of Intein-Mediated Protein Splicing. In Nucleic Acids and Molecular Biology16 (Homing Endonucleases and Inteins), ed. M. Belfort, 231-255, Berlin: Springer Verlag.
8. Mills, K.V., *Manning, J.S., *Garcia, A.M., *Wuerdeman, L.A. (2004) Protein splicing of a Pyrococcus abyssiintein with a C-terminal glutamine.  J. Biol. Chem.279, 20685-20691.
7. Mills, K.V. (2004) Self-Splicing Proteins.  In: Handbook of Proteolytic EnzymesSecond EditionVol. 2., ed. AJ Barrett, 2136-2140, London: Academic Press, Elsevier Science.
6. Mills, KV.  (2003) Post-Translational Control.  In: Genetics, Vol. 3., ed. R Robinson, 178-182, New York: Macmillan Reference USA.
5. Mills, K.V., Paulus, H.  (2001) Reversible Inhibition of Protein Splicing by Zinc Ion.  J. Biol. Chem. 276, 10832-10838.
4. Lew, B.M., Mills, K.V., Paulus, H. (1999) Characteristics of Protein Splicing in trans Mediated by a Semisynthetic Split Intein. Biopolymers (Peptide Science)51, 355-362.
3. Lew, B.M., Mills, K.V., Paulus, H. (1998) Protein Splicing in Vitro with a Semisynthetic Two-Component Minimal Intein. J. Biol. Chem.273, 15887-15890.
2. Mills, K.V., Lew, B.M., Jiang, Sq, Paulus, H.  (1998) Protein splicing in trans by purified N- and C- terminal fragments of the Mycobacterium tuberculosis RecA intein. Proceedings of the National Academy of Sciences, USA95, 3543-3548.
1. Broomfield, C.A., Mills, K.V., Meier, B.M., Lockridge, O.A., and Millard, C.B.  (1998) Reaction of human butyrylcholinesterase (BchE) H117 enzymes with carbamates.  In: Structure and Function of Cholinesterases and Related Proteins, 223-226.

Ken Mills, Ph.D., Principal Investigator

Barbara Aikaterinidis, 2024
Cyndie Alexis, 2025

Bea Barbesino, Chemistry 2023

Anthony D'Angelo, Chemistry 2022
Brynn Dundon, 2025
Michelle Haigbea, Biology 2023
Lily Nguyen, Chemistry/Sociology 2024

Thuy Nguyen, Chemistry/Sociology 2022
Kallie Poon, Biology 2024
Lily Russo, Chemistry 2022
Diana Wall, Biology 2022

Josue Alvarado, Sociology 2023
Justin Hackshaw, Economics 2022

2021
Angel Chavez, Chemistry, Pfizer
Bella Jankowski, Chem/Biochem, PhD student, UMass
Mercedes Ruiz, English
Sean Tarrant, Bio/Biochem, Clinical Research Coordinator
Vaish Yalala, Chemistry, Clinical Research Coordinator

 
2020
Alexandra Cawood, Chem/Biochem, Clinical Research Coordinator
Chinnel Hudson, Sociology


 2019
Owen Conroy, Biology, Broad Institute
Patrick Exconde, Chem/Biochem, PhD student UPenn
Abby Lynch, Biology/Biochem,
Clinical Research Coordinator
Nhi Tran, UMass Amherst
Amanda Wibben, Biology Fulbright India, Theology/Ethics grad student Harvard


 2018
Hannah Comeau, Chemistry, Masters, Johns Hopkins, Clinical Research Coordinator
Vanessa Costa, Sociology, Clinical specialist
C.J. Janton, Bio/Biochem, DO/PhD student, NYIT
Christian Ramsoomair, Chem/Biochem MD/PhD student, Miami
Deirdre Reidy, Biology, MD student, UConn


2017
Shirley Amunya, Fitchburg St, Teach for America
Alexander Bonano, Spanish, MPH student, Tufts
Josh Long, Biology, MD Student
Chris Minteer, Chem/Biochem PhD student, Yale
Wanlin Zhang, Chem/Biochem, Dental student, Touro


2016
Chris CousinChemistry, PharmD, MCPHS
Julliard del Rosario, Cornell U
Mike Koulopoulos, Chem/Biochem, MD Wake Forest
Anthony Yakely, Chem/Biochem, JVC Northwest, MPH Yale


  2015
Dawilmer Castillo, Bridgewater St., Masters, Brown, Pharmacist
Zach Giaccone, Chem/Biochem, Dental School, UConn
Mario Jaramillo, Biology, MD, Albany Med
Isabelle Pierre, UMass Dartmouth
Nikki Siegart, Bio/Biochem, MD SUNY Downstate


  2014
Sarah Cahn, Chemistry, Takeda Oncology
Dan Haley, Chemistry DDS, UConn


  2013
Katie Colelli Chiarolanzio, Bio/Biochem, M.D., UVM
Nick Gulbrandsen, Bio Fitchburg
Michael Nicastri, Chem/Biochem PhD, UPenn, PD Columbia
Jen Pusztay, Chem/Biochem , D.O. UNECOM
Laura Urbanski , Chem/Biochem MD/PhD program, UConn
Jennie Williams, Chemistry , Teacher, Chapel Hill-Chauncy Hall


  2012
Stacy Chin, Chemistry, PhD, Boston U
Kevin Karanja, Chemistry WSU, Science Teacher
George Savidis, Bio/Biochem, Cytivia


  2011
Colleen Donahue,Biology, M.D., Tufts
Michelle Marieni, Biology, M.D., Creighton
Kevin O’Brien, Bio/Biochem PhD, U. Georgia, PD UMass Med
Kristina Xega, Chemistry, Takeda Oncology


  2010
Lauren Duffee, Bio/Biochem, D.V.M., UPenn
Adam Nadelson, Biology, M.D., UMass
Dan York, Bio/Biochem, MD, Texas Tech


  2009
Adam Kerrigan, Biology Cybereason
Annie Schufreider, Chemistry, M.D., Loyola Chicago
Jen Winslow, Biology, DO, University of New England


  2008
Melissa McGill, Bio/Biochem, M.S., Michigan, Pfizer
Kathryn O’Brien, Chem/Biochem PhD, Cornell-Weill


  2007
Matt Drago,Bio/Biochem, M.D., UPenn
Taryn Powers Surabian, Biology Ursuline Academy


  2006
Deirdre Dorval Wells, Biology J.D., Harvard Law School


  2005
Katie Lewandowski Bagnowski, Bio/Biochem PhD, Wash. U., PD Northwestern


  2004
Kate Leahy, Math, MD, Boston U
Jackie Gareau Merrick, Chem/Biochem, Ph.D, Cornell-Weill
Tim Quinn, Chem/Biochem, M.D.-Georgetown


  2003
Jenny Manning Chapnick, Biology
Lisa Wuerdeman Lucas, Chemistry D.O., U. New England
Ralph Millilo, Bio/Biochem, M.D. SUNY Upstate
Lisa Garcia Seals, Biology Middle School Teacher


  2002
Beth Ambrose Dean, Chemistry Physician Assistant
Pat Twomey, Chem/Biochem, M.D., Georgetown


Kate Connor, Research Assistant
Alvin Gomez, Research Associate 
Julie Reitter, Ph.D., Research Associate 

Mills Lab Photos