Mike Kent

Biography

Mike Kent began his career at Sandia National Labs as a materials scientist studying polymers, thin films, and interfacial physical and chemical phenomena. Kent then moved to the Biological and Materials Science Center at Sandia to research cellulose and lignin breakdown using catalysts and enzymes. He has also studied protein structure, enzymology, antibiotics, viruses, and therapeutic antibodies. He has extensive experience with vibrational spectroscopy (FTIR); fluorescence/FRET; enzyme assays; surface/interfacial analysis tools such as AFM, SIMS, XPS, ellipsometry, and quartz crystal microbalance with dissipation monitoring (QCM-D); and neutron and X-ray scattering, reflection, and diffraction. With his expertise, leadership, training, and motivation, Kent has successfully administered many projects (e.g. staffing, research directions, budget), collaborated with other researchers from a wide array of institutions, and produced peer-reviewed publications from each project.

Education

• Bachelor’s Degree: Chemical Engineering, University of Illinois, Urbana- Champaign (1979-1984)
• Doctoral Degree: Chemical Engineering and Material Science, University of Minnesota (1984-1990); Advisors: Matthew Tirrell and Timothy Lodge
• Postdoctoral Fellowships: Polymer Physics, Curie Institute, Paris, France (1990-1992); Advisor: Francis Rondelez

Relevant Positions

• Research Scientist, Materials Research Center, Sandia National Laboratories (1992-2005)
• Research Scientist, Biological and Materials Science Center, Sandia National Laboratories (2005-Present)
• Research Scientist, Joint Bioenergy Institute (2007-Present)

Research Interests

Interaction of Envelope Protein of Dengue Virus with Lipid Membranes

Enveloped viruses use a protein hairpin conformational change to mediate fusion with host cell membranes, but the biophysical mechanisms are not understood.  Understanding this complex process could aid in the development of fusion inhibitors. Kent recently led a 3 year project to study the interaction of the envelope protein of Dengue virus with lipid membranes.  This program combined molecular simulations with data from neutron reflectivity (NR), quartz crystal microbalance, and a new technique we developed to measure the anchoring energy of strongly-bound membrane-associated proteins. The envelope (E) protein of Dengue virus forms a trimer that mediates fusion of viral and host membranes.  Insertion of the tip of E into host membranes is essential to the process, serving to anchor E into the membrane and possibly also to disrupt local ordering within the membrane and thereby initiate membrane fusion.  Both aspects are impacted by the depth of insertion, the orientation of the trimer with respect to the membrane normal, and the interactions that form between protein and membrane. Experimental and simulation results showed that E inserts at an angle with respect to the membrane due to electrostatic interactions between positively-charged lysines on the sides of the trimer and the lipid headgroups.  The results also showed that the tip of E imbeds into the interface between the headgroups and acyl chains of the outer leaflet, and that tilting of E disturbs the lipid ordering around the inserted tip. Kent directed this program, performed the NR and QCM studies, and led development of a new biophysical method to measure the energy to unbind strongly-bound membrane-associated proteins from the membrane.

Relevant Publications:

• Vanegas, JM, Heinrich, F, Rogers, DM, Carson, BD, La Bauve, S, Vernon, BC, Akgun, B, Satija, S, Zheng, A, Kielian, M, Rempe, SB, Kent, MS (2018).  Insertion of Dengue E into Lipid Bilayers Studied by Neutron Reflectivity and Molecular Dynamics Simulations. BBA – Biomembranes 1860, 1216-1230.  Pubmed: 29447917
• La Bauve, S, Vernon, BC, Ye, D, Rogers, DM, Siegrist, CM, Carson, BD, Rempe, SB, Zheng, A, Keilian, M, Shreve, AP, Kent, MS.  (2016). Method for Measuring the Unbinding Energy of Strongly-Bound Membrane-Associated Proteins.  BBA – Biomembranes 1858, 2753-2762. Pubmed: 27425029
• Rogers, DM, Kent, MS, Rempe, SB (2015). Molecular Basis of Endosomal-Membrane Association for the Dengue Virus Envelope Protein.  BBA – Biomembranes 1848, 1041-1052. Pubmed: 25559317

Methods to Study the Conformations of Membrance-Associated Proteins

Kent recently developed methods to study the conformations of membrane-associated proteins and applied these methods to membrane-bound HIV-1 Nef.  While the membrane plays an essential role in the structure and regulation of many proteins, unfortunately membranes interfere with mainstream structural analysis methods such as crystallography and NMR.  Therefore, information that can be obtained for membrane-associated proteins at present is very limited (i.e. secondary structure by CD or FTIR, positioning of specific labeled residues by FRET or EPR) and is labor intensive and time consuming to generate. The new approach, developed in collaboration with Professor John Engen (Northeastern U.), combines neutron reflectometry with hydrogen-deuterium exchange mass spectrometry (HDX-MS) for proteins associated with Langmuir monolayers.  NR provides the in-plane averaged residue distribution normal to the membrane and HDX-MS provides local information about solvent accessibility and dynamics of specific peptides and motifs.  HIV-1 Nef is critical to AIDS progression and functions in the membrane-associated state as an adaptor protein to alter signaling, trafficking of immune and viral receptors, and enhance viral replication.  Our NR work provided the first structural data for membrane-bound Nef, showing that in absence of a host protein Nef transitions from a closed to an open form as a function of membrane properties (packing density or membrane pressure) where the core domain is displaced 70 Å from the lipid membrane.  The HDX-MS studies showed i) that a critical flexible loop of Nef that contains a dileucine sorting motif is more protected from H/D exchange in the membrane-bound state than when free in solution, and ii) that Nef does not dimerize on its own (in absence of host proteins). Kent led the NR studies and conceived of the methods to perform the H/D exchange for the membrane-bound protein in a Langmuir trough and capture the surface layer for MS studies of the rate of exchange.  He performed the initial studies and the work was then transferred to a graduate student in the Engen lab to complete detailed studies.  Two publications resulted from the NR studies and two publications resulted from the new HDX-MS methodology.

This methodology will provide conformational information for integral and peripheral membrane proteins at moderate-level resolution along with local information in the form of solvent accessibility along the entire length of the protein.   This combination of methods will allow measurement of size, shape, dynamics, positioning of protein domains relative to the membrane, and conformational changes for proteins in their membrane-associated conformations.  The methods will be applicable to many integral and peripheral membrane proteins that are not amenable to other structural methods, such as crystallization or NMR. While this approach will eventually have broad application to a wide range of proteins, a particular class that will be immediately impacted is the large group of proteins (hundreds) that are modified by lipid groups for targeting to the lipid membrane.  Many of these proteins are involved in signaling and require membrane association to signal efficiently.  In some cases membrane association through the lipid moiety causes a dramatic conformational change of the protein from cytosolic to membrane-bound form.  For example, myristoyl or farnesyl switch mechanisms are known for more than a dozen proteins, with Arf GTPase being a hallmark example. These mechanisms promote membrane binding, facilitate release from the membrane-bound state, and regulate protein-protein interaction.

Relevant Publications:

• Akgun, B., Satija, S., Nanda, H., Pirrone, GF, Shi, X., Engen, JR., Kent, MS (2013). Conformational Transition of Membrane-Associated Terminally Acylated HIV-1 Nef. Structure, 21,1822-1833.  Pubmed: 24035710
• Kent MS, Murton JK, Sasaki DY, Satija S, Akgun B, Nanda H., Curtis JE, Majewski J, Morgan CR, Engen, JR (2010). A Study of the Conformation of HIV Nef Bound to Lipid Membranes by Neutron Reflectometry.  Biophysical Journal 99, 1940-1948. Pubmed: 20858440
• Pirrone, GF, Vernon, BC, Kent, MS, Engen, JR (2015). Hydrogen Exchange Mass Spectrometry of Proteins at Langmuir Monolayers.  Analytical Chemistry 87, 7022-7029. Pubmed: 26134943
• Pirrone, GF, Emert-Sedlak, LA, Kent, MS, Wales, TE, Smithgall, TE, Kent, MS, Engen, JR (2015). Membrane-associated Conformation of HIV-1 Nef Investigated with Hydrogen Exchange Mass Spectrometry at a Langmuir Monolayer. Analytical Chemistry 87, 7030-7035. Pubmed: 26133569

Collaborative Work with the Joint BioEnergy Institute- Lignin Studies

In prior work in association with the Joint BioEnergy Institute (JBEI), our team combined experimental measurements of enzyme activity and quantum chemistry calculations of free energy changes of relevant chemical reactions and of electron spin density distributions of radical species to understand the selective oxidation of phenolic versus non-phenolic lignin model compounds by a versatile peroxidase.  This work also revealed that selection between polymerization and depolymerization reaction pathways depends on the functional group at the 5 position of the guaiacyl group.  This work resulted in a publication in Green Chemistry that was highlighted on the cover and selected for the 2017 HOT Articles Collection by this journal.

Also in further work associated with JBEI, I developed a new multiplexed assay for lignin degrading enzymes and catalysts.  I used this method to optimize the conditions for oxidative chelator-mediated Fenton reactions to convert insoluble lignin into a water-soluble polymer that functions as a dispersant, and to optimize reactions involving laccases combined with low molecular weight mediators.

Relevant Publication:

• Zeng, J, Mills, MJL, Simmons, BA, Kent, MS, Sale, KL (2017).  Understanding factors controlling depolymerization and polymerization in catalytic degradation of b-ether linked model lignin compounds by versatile peroxidase.  Green Chemistry 19, 2145-2154.
• Kent, MS, Avina, IC, Rader, N, Busse, ML, George, A, Sathitsuksanoh, N, Baidoo, E, Timlin, J, Giron, NH, Celina, MC, Martin, LE, Polsky, R, Chavez, VH, Huber, DL, Keasling, JD, Singh, S, Simmons, BA, Sale, KL (2015).   Assay for lignin breakdown based on lignin films:  insights into the Fenton reaction with insoluble lignin.  Green Chemistry 17, 4830-4845.
• Kent, MS, Zeng, J, Rader, N, Avina, IC, Simoes, CT, Brenden, CK, Busse, ML, Watt, J, Giron, NH, Alam, TM, Alendorf, MD, Simmons, BA, Bell, NS, Sale, KL (2018).   Efficient conversion of lignin into a water-soluble polymer by a chelator-mediated Fenton reaction:  optimization of H₂O₂ use and performance as a dispersant.  Green Chemistry in press.

Synergistic Activity of Cellulase Cocktails for Biomass Deconstruction

Kent has also performed a series of studies on the synergistic activity of cellulase cocktails for biomass deconstruction.  Cellulase cocktails include exoglucanases that digest from the ends of cellulose chains and endoglucanases that cleave randomly at interior points along cellulose chains. In addition, cellulose binding domains (CBDs) are known to play an important role in the digestion of crystalline cellulose but much less is known about the benefit of CBDs in the digestion of amorphous cellulose. Amorphous cellulose is of interest as it results from ionic liquid pretreatment of biomass, a promising second generation pretreatment technology. In collaboration with coworkers at JBEI, Kent combined studies of structural changes in cellulose films during digestion by neutron reflectivity, measurements of changes in mass and film stiffness using a quartz crystal microbalance, and visualization of the motion of individual enzymes by total internal reflectance (TIRF) microscopy to unravel the role of endoglucancases and cellulose binding domains in enhancing the activity of exoglucanses on amorphous cellulose.  He performed the NR, QCM, and TIRF studies, and directed the other experimental work.  This work showed that certain cellulose binding domains drive the penetration of enzymes into amorphous cellulose.  This work supports a large enzyme engineering effort to couple CBDs with catalytic domains to design an enzyme cocktail optimized for ionic-liquid treated cellulose.

Relevant Publication:

• Cheng, G., Datta, S., Liu, Z, Wang, C., Murton, JK, Brown, PA, Jablin, MS,  Dubey, M, Majewski, J, Halbert, CE, Browning, JF, Esker, AR, Watson, BJ, Zhang, H., Hutcheson, SW, Huber, DL, Sale, KL, Simmons, BA, Kent, MS (2012) Interactions of endoglucanases with amorphous cellulose films resolved by neutron reflectometry and quartz crystal microbalance with dissipation monitoring.  Langmuir 28, 8348-8358.

Neutron Reflectivity to Study Structures of Polymers at Interfaces

Early publications focused on the use of neutron reflectivity to study the structures of polymers at interfaces.  Block copolymers are surfactants that can be used to stabilize polymer blends or particle dispersions.  In a series of publications, Kent used NR to characterize the segment distribution of block copolymers for which one (small) block was adsorbed to an interface and a second (large) block remained in solution.  The stabilizing ability of these materials is determined largely by the segment density profile in the interfacial layer.  He measured the detailed segment density profiles for a wide variety of conditions and compared with theoretical predictions.  He conceived of and performed all the NR studies.

Relevant Publication:

• Kent MS. (2000).  A quantitative study of tethered chains in various solution conditions using Langmuir diblock copolymer monolayers. Macromolecular Rapid Communications 21, 243-270.

Publications

Throughout his career, Kent has published 74 articles on his research.

Selected News Article

‘Researchers at Sandia, Northeastern develop method to study critical HIV protein’ Article by Mollie Rappe (August 3, 2016).

Honors and Memberships

• Alpha Chi Sigma Scholarship Plaque (1981)
• Scholarship Award, Eastman Kodak Company (1982)
• Scholarship Award, Union Oil Company (1983)
• Student Award of the American Institute of Chemists (1984)
• Graduated Summa Cum Laude, University of Illinois (1984)
• National Science Foundation Fellowship (1985-1988)
• Member, Program Advisory Committee, Los Alamos Neutron Science Center (1999-2001)
• Member, User Group Executive Committee, Los Alamos Neutron Science Center (2001-2003)
• Member, Program Advisory Committee, NIST Center for Neutron Research (2001-2009)
• Member, Biophysical Society (2004-Present) Member, Neutron Scattering Society of America (2002-Present)
• Program Co-Chair, American Conference on Neutron Scattering (1985-1988)