Education

Ph.D. Oregon State University (Biochemistry-Biophysics) 1984

M.S. University of Chicago (Biochemistry) 1977

A.B. Ripon College (Chemistry) 1976

Professional Experience

Researcher, Student Originated Studies, Ripon College, Ripon, Wisconsin 1974

Undergraduate Research Trainee, Holifield National Laboratories, Oak Ridge, Tennessee 1975

Research Assistant, Michael Reese Hospital - Renal Division, Chicago, Illinois 1977-1978

Postdoctoral Researcher, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 1983-1987

Postdoctoral Researcher, University of Wisconsin-Madison, Madison, Wisconsin 1987-1990

Organizer and Chairman, Madison Minisymposium on Staphylococcal Nuclease. August 20-21, 1988

Assistant Professor, Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,Ohio State University 1990-1997

Visiting Assistant Professor, Department of Chemistry, Ohio Northern University, Ada, OH 1997-1998

Assistant Professor, Chemistry Department, Black Hills State University, Spearfish, SD 1998-Present Professional Societies (member since)

American Chemical Society (1983)

Sigma Xi (1990)

Compact Domains

In this project both theoretical and experimental studies are being performed to identify domain, subdomain, and sub-subdomain structure in proteins. My interest in this field derives from the fact that protein domain and subdomains play an important role in the protein folding pathway, and may be key intermediates in de novo protein design.

My work in this area started several years ago while I was a postdoctral fellow with Dr. George Rose. In this work I showed that a protein's continuous domains could be found by evaluating the compactness of continuous substructures within the protein. There were two problems with this work, however. First, the approach could not be extended to find discontinuous domains because of the large amount of computer time required for calculations of compactness. Second, since Dr. Rose's group did only theoretical work, there was no way to provide the experimental proof that the compact domain theory actually meant something.

In my compact domain project I have been able to successfully attack both of these problems directly. On the theoretical side I have created a new calculation method that allows me to do calculations of compactness thousands of times faster than before, and this has allowed me to use compactness to locate binary discontinuous domains in proteins of known structure. I have currently developing a new algorithm that allows me to locate domains containing any number of discontinuous polypeptide chains.

Future Work

I am also very interested in collaborations. I have used a wide range of instrumentation, and I am always interested in trying to use this instrumentation to help somebody out on a problem. I think collaborations are particularly important in smaller schools, both because they can make a limited budget stretch a little farther, and because the students need to see that a given problem may be attacked in several different ways. I am interested in talking with anybody to see if there are any areas of mutual interest

Isosteres in the Alpha Helix

Isosteres are alternate ways of connecting amino acids in peptide mimetics. The naturally occurring peptide bond is biologically labile, so isosteres are frequently used to give peptide mimetic drugs a longer lifetime in the bloodstream. The structural implications of replacing a peptide bond with an isostere are not well understood, so one aim of this project is to analyze how an well an isostere can be incorporated into a common piece of protein structure, the alphahelix. The real beauty of this project is that this same line of experimentation can answer an important basic question of protein structure that has remained unsolved for 40 years.

The strength of the hydrogen bond in a helix has been debated in the literature since Pauling first proposed the alpha helix structure in 1951. The debate has raged over such basic issues as whether the backbone hydrogen bond is favorable or unfavorable (+ or - ) delta G or whether it is primarily entropic (delta S) or enthalpic (delta H) in nature.

In this project a series of isosteres are placed in a model alpha helical peptide. Using circular dichroism (CD) we can analyze whether or not the peptide remains helical, and this tells us directly if a given isostere can be incorporated into a helix. Further, if we carefully analyze the melting curves of these peptides we can derive the delta G and delta H of the helix to coil transition. When the delta G and delta H of the normal peptide and the isosterically linked peptide are compared we can then determine the delta-delta G and delta-delta H for the structural change of peptide linkage6isosteric linkage. By incorporating an isostere that selectively removes a single hydrogen bond this system allows us to directly measure the strength of the hydrogen bond and resolve an important 40 year old problem.

Preliminary work in this area has focused on the N-methyl isostere because it is easy to incorporate in a standard F-moc synthesis. We have discovered that this peptide is inimical to the helix; it almost completely destroys the helix in every peptide we have tried. This effect goes well beyond what we expected for the removal of a hydrogen bond and it is likely that there are additional stearic interactions between the bulky methyl group and adjacent amino acids that contribute additional destabilization. Since there are several different interactions that could be effected, we are presently trying to characterize this system more precisely.

This project is ideal for undergraduate research. Right now I have two undergraduates working to synthesize some of the required isosteres. Over the next couple of summers, I will have students incorporating these isosteres into models helixes, purifying the helical peptides using HPLC, and then characterizing the helices using CD.

Research

Presently there are two major research efforts in my lab. The first deals with determining the strength of a backbone hydrogen bond in an alpha helix, and the second deals with determining the thiamine levels (vitamin B1) in various tissues of walleye and salmon taken from Lake Oahe in central South Dakota. I have also done theoretical work on protein domains. At this present time the work on the alpha helix is supported through a grant from the National Science Foundation, and the thiamine studies are done in collaboration with State of South Dakota Game Fish and Parks - McNenny Fish Hatchery with funds from the Federal Sport Fish Restoration Fund

Research Background

My graduate degree was earned at Oregon State University where I worked with Dr. W. Curtis Johnson, Jr. In this work I used a wide range of biophysical methods to determine the exact nature of the P-form DNA, a structural form of DNA that exists in alcohol solutions. I was eventually possible to prove the P-from was a combination of collapsed tertiary structure and denatured secondary structure. The presence of collapsed tertiary structure was indicated in viscosity experiments and proven by direct visualization via electron microscopy, while the denatured secondary structure was first indicated by a delayed P-form transition in covalently-closed circular DNA and proven directly through infrared spectroscopy.

My first post-doctoral position was with Dr. George Rose, then at the Pennsylvania State University - Hershey Medical School. In this work I developed a new method for identifying domains and subdomains in proteins. While the presence of domains has been a central feature in our understanding of protein structure for many years, a satisfying definition of domain structure based on rigorous mathematical criterion and sensible physical properties has been lacking. This work defined domains as continuous pieces of the polypeptide chain having a minimum surface area for their enclosed volume. This definition is mathematically exact, physically reasonable, and yields units which agree well with one's intuitive expectations.

My second post-doctoral position was with Dr. John Markley at the University of Wisconsin. Here I worked on two major NMR projects. In the first, I used standard 1H 2D NMR techniques to determine the solution structure of a DNA octamer in the presence of spermine, and tried to determine if spermine was bound to the DNA molecule. In the second project I learned various 2-D and 3-D heteronuclear NMR techniques in an attempt to assign completely all resonances (1H, 13C, and 15N) for a ferrocytochrome c553. Besides being used to help determine the structure of the protein, the data accumulated in this project were used to help develop computer-based strategies for assignment of resonances, and to determine the usefulness of new NMR experiments.

Thiamine in Fish

Eggs from Salmon spawned from Lake Oahe fish usually perform poorly in the hatchery, with only 20-30% of the eggs ever developing to become fish that can be replanted in the lake. Many studies in the literature have linked poor egg survivability with low levels of thiamine levels found in the eggs of these fish.

We started this study in 1999 to see if we could document thiamine levels in Lake Oahe Salmon, and to see if we could add thiamine to eggs prior to their incubation, to increase their survivability in the hatchery. Our results proved to be quite puzzling; near normal levels of thiamine were found, and egg survivability was exceptionally good that year with or without our added thiamine treatments. The answer to this puzzle was found in population studies done by the Game Fish and Parks.

Their studies showed that in 1999 the smelt population (the normal prey of the Salmon) was exceptionally low, so the Salmon were feeding on other fish and insects instead of their normal smelt prey. Why should this affect the salmon thiamine levels? The smelt are thought to contain an enzyme called thiaminase that breaks down thiamine. As a result, as salmon eat the smelt, they get the thiaminase in their gut, where it breaks down any thiamine they have ingested, and leaves the fish thiamine deficient. have large amounts of this thiaminse in their digestive tract, and this destroys any thiamine.

Walleye are another fish in Lake Oahe that feeds on the smelt, and the success rate of walleye spawning suggests that they too, might be suffering thiamine deficiencies. As a result, our study has been expanded to measure thaimine levels in both walleye and salmon taken from various locations around the lake. Beginning in the summer of 2001 we will also begin measuring thiaminase levels in smelt found in Lake Oahe to see if we can tie the entire system together.

Long term correlations of this data with Fisheries data on reproductive success and overall fishing success of these fish in Lake Oahe is expected to have a major impact on fish management in Lake Oahe over the next several years.

Abstracts

M. H. Zehfus and G. D. Rose, Location and Evaluation of Protein Domains, Biophysical Journal 47, 181a, (1985).

M. H. Zehfus and G. D. Rose, Compact Domains in Proteins, Biophysical Journal 49, 290a, (1986).

M. H. Zehfus, Discontinuous Compact Protein Domains, Protein Science 2, Supplment 1, 68, (1993).

M. H. Zehfus, Unitary and Binary Compact Domains in Proteins, Biophysical Journal 66, A14, (1994).

M. H. Zehfus, Automatic Identification of Hydrophobic Clusters, Biophysical Journal 68, A250 (1995).

M. W. Maciejewski and M. H. Zehfus, Structure of a Compact Peptide Determined by Circular Dichroism and NMR, Biophysical Journal 68, A325, (1995).

C.-F. Chang and M. H. Zehfus, Effects of N-Methyl Isosteres on Helix Stability, 14th American Peptide Symposium Program and Abstract, P493, (1995).

M. W. Maciejewski and M. H. Zehfus, Structure of a Compact Peptide, Residues 99-141 of Staphylococcal Nuclease, Determined by Circular Dichroism and NMR, Protein Science 4, Suppl.2, 51-S, (1995).

M. H. Zehfus, Automatic Identification of Hydrophobic Clusters, Protein Science 4, Suppl.2, 83-T, (1995).

M. W. Maciejewski and M. H. Zehfus, Circular dichroism and NMR analysis of a compact peptide (99-140) from staphylococcal nuclease in both aqueous and 40% 2,2,2-triflourothanol solutions, Biophysical Journal 70, A105, (1996).

M. H. Zehfus, Locating Discontinuous Compact Domains Containing more than Two Peptide Units, Biophysical Journal 70, A444, (1996).

M. H. Zehfus, Identifying Compact Domains with more than Two Peptides, Protein Science 5, Suppl. 1, 44-S, 66 (1996).

C.-F. Chang and M. H. Zehfus, Effects of N-methyl Isosteres on Helical Peptides, Protein Science 5, Suppl. 1, 90-S, 74 (1996).

C.-F. Chang and M. H. Zehfus, The Effect of Peptide Backbone modification on Helix Structure, Biophysical Journal 72, A393, (1997).

K. S. Stock, F. Farrokhi, J. A. Shumacher, R. L. Nutter, M. Zehfus, M. Barnes, Measuring Thiamine Levels in Lake Oahe Salmon, Abstract Book - 14th National Conference on Undergraduate Research 39 (2000).

Publications

T. H. Broman, D. W. Carlson, M. D. Milikowski, M. H. Zehfus, and D. J. Minima, Student Originated Study Abstract Reports, 69-70, (1974).

P. J. Daugherty, N. K. Clapp, M. H. Zehfus, and S. E. Brock, GANN 68(5), 697-702, (1977).

M. H. Zehfus and W. C. Johnson, Jr., Properties of P-form DNA as Revealed by Circular Dichroism, Biopolymers 20, 1589-1603, (1981).

M. H. Zehfus and W. C. Johnson, Jr., Conformation of P-form DNA, Biopolymers 23, 1269-1281, (1984).

M. H. Zehfus, J. P. Seltzer, and G. D. Rose Fast Approximations for Accessible Surface Area and Volume of Protein Segments, Biopolymers 24, 2511-2518, (1985).

G. D. Rose, A. R. Geselowitz, G. J. Lesser, R. H. Lee, and M. H. Zehfus, The Hydrophobicity of Amino Acid Residues in Globular Proteins, Science 299, 834-838, (1985).

G. J. Lesser, R. H. Lee, M. H. Zehfus, and G. D. Rose, Hydrophobic Interactions in Proteins, Protein Engineering, New York: Alan R. Liss Inc., (1987).

M. H. Zehfus and G. D. Rose, Compact Units in Proteins, Biochemistry 25, 5759-5765, (1986).

M. H. Zehfus, Continuous Compact Protein Domains, Proteins: Structure, Function and Genetics 2, 90-110 (1987).

M. H. Zehfus, M. D. Reily, E. L. Ulrich, W. M. Westler, and J. L. Markley, 1H, 13C, and 15N Resonance Assignments for a Ferrocytochrome c553 Heme by Multinuclear NMR Spectroscopy. Archives of Biochemistry and Biophysics 276, 369-373 (1990).

M. H. Zehfus, Improved Calculations of Compactness and a Re-evaluation of Continuous Compact Units. Proteins: Structure, Function and Genetics 16, 293-300 (1993).

M. H. Zehfus, Binary Discontinuous Compact Protein Domains Protein Engineering 7, 335-340 (1994).

M. H. Zehfus, Automatic Recognition of Hydrophobic Cluster and their Correlation with Protein Folding Units. Protein Science 4, 1188-1202 (1995).

M. W. Maciejewski & M. H. Zehfus, Structure of a Compact Peptide from Staphylococcal Nuclease Determined by Circular Dichroism and NMR Spectroscopy. Biochemistry 34, 5795-5800 (1995).

C.-F. Chang & M. H. Zehfus, Effects of N-Methyl Isosteres on Helix Stability, Proceeding of the 14th American Peptide Symposium (Peptides: Chemistry, Structure and Biology, PTP Kaumaya & RD Hodges Eds. Escom Leiden, The Netherlands, 1995).

M. W. Maciejewski & M. H. Zehfus, 2,2,2-Trifluoroethanol Induced Structural Change in a Compact Domain from Staphylococcal Nuclease, Undergoing revision for Protein Science

M. H. Zehfus, Identification of compact, hydrophobically stabilized modules and domains containing one or more peptide chains. Protein Science 6, 1210-1219 (1997).

C.-F. Chang & M.H.Zehfus, The effect of N-methylation on helical peptides. Biopolymers 40, 609-616 (1997).

C.-F. Chang & M. H. Zehfus, The Effect of Backbone Modification of Helical Peptides: The Reduced Carbonyl Modification. Biopolymers 46, 181-193 (1998).

Barnes, M. E., M. H. Zehfus, F. Farrohki, R. L. Nutter, J. A. Schumacher, and K. S. Stock. Initial observations on the use of thiamine hydrochloride treatments during land-locked fall chinook salmon spawning. North American Journal of Aquaculture, In Press.

Grants

Federal Aid Study #1587 , "Thiamine Influences on Lake Oahe Walleye Reproduction and the Hatchery Survival of Walleye and Chinook Salmon Embryos Obtained as Eggs from Lake Oahe Broodstock." 5/5/00-5/4/05. $138,900 Mike Barnes is PI, I am Co PI.

National Science Foundation - Division of Molecular and Cellular Biosciences, MCB-9986235, "Determination of Hydrogen Bond Strength in an Alpha-Helix by Replacement of Backbone Functional Groups." 6/15/00-5/31/03. $112,935. I am PI

National Institutes of Health Postdoctoral Fellowship, National Institute of General Medical Sciences GM10824 - "Location, Evaluation, and Taxonomy of Protein Domains." 6/1/85 - 4/30/86.

National Institutes of Health FIRST Award, National Institute of General Medical Sciences R29 GM46664 "Compact Domains in Proteins." 5/1/92 - 8/31/97. Total direct costs for period - $350,000.

Awards

Graduated Magnum Cum Laude, Ripon College (1976)

Phi Beta Kappa (1976)

Phi Lambda Upsilon (1982)

Rho Chi (1991)