Email: j.manning@neu.edu

Phone: 617.373.5267
Fax: 617.373.4496

Location: 405 Mugar Life Sciences
Mail: NU/Biology
         134 Mugar Life Sciences
         360 Huntington Avenue
         Boston, MA 02115 USA

Research Description

Our goal is to further our understanding of proteins, especially those that have important biological functions. We employ both the classical methods of protein chemistry as well as the newer recombinant DNA technology.

Hemoglobin - In order to locate the functional chloride binding sites in hemoglobin, we employed a novel strategy of random chemical modification. Molecular modeling indicated that several of these sites are aligned along the central dyad axis of the molecule to form a chloride channel connecting the major binding sites at either end of the central dyad axis. It is likely that they function by keeping the central dyad axis in the open mode in deoxyhemoglobin and thus modulate the ease with which oxygen is released. The a- and b-globin genes of human sickle hemoglobin constructed on a single plasmid have been expressed in yeast. The recombinant sickle hemoglobin is identical to natural human sickle hemoglobin in every property tested thus far. A recombinant double mutant sickle hemoglobin has recently been expressed in this system with Isoleucine replacing an exterior Lysine; there is a very large inhibitory effect in gelation.

Acylpeptide Hydrolase - The biological role of this enzyme may be to remove the acetyl group together with the first amino acid of nascent polypeptide chains as a co-translational event during protein biosynthesis. The enzyme also acts on N-blocked bioactive peptides. We have recently identified the active site Serine and Histidine residues in the catalytic triad of this protease. Acylpeptide hydrolase belongs to the new class of proteases, the hydrolase-fold family.

Bacterial D-Amino Acid Transaminase - This bacterial enzyme is responsible for the synthesis of D-amino acids in the bacterial cell wall. Its exclusive presence in bacteria endows it with intrinsic specificity since it is not present in mammalian cells, which do not require D-amino acids. We expect that specific inhibitors for this enzyme will be developed and that these could be novel antimicrobial agents. Site-directed mutagenesis techniques together with a complete kinetic analysis of the catalytic steps of the mutant enzymes are being used to study its mechanism. When exposed to D-alanine, the normal substrate for this enzyme, for a period of hours the enzyme is inactivated by a mechanism in which a reactive intermediate undergoes non-productive decarboxylation and immobilizes the coenzyme in one of the two subunits. This phenomenon may be an example of protein ageing.

Lois R. Manning, J. Eric Russell, Julio C. Padovan, Brian T. Chait, Anthony Popowicz, Robert S. Manning, and James M. Manning.  Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation withlifespan in the red cell. Protein Science, 16:1641 - 1658 (2007).

M. Ashiuchi, T. Yagami, R.J. Willey, J.C. Padovan, B.T. Chait, A. Popowicz, L.R. Manning, and J.M. Manning. N-Terminal Acetylation and Protonation of Individual Hemoglobin Subunits. Protein Science, 14:1458 - 1471 (2005).

J.M. Manning
Acylaminococyl-peptide (Acylpeptide Hydrolase) Handbook of Proteolytic
Enzymes, 2nd Ed.
Elsevier, pgs. 1917 - 1919 (2004)

A.Geva, J.J. Clark, Y.Zhang, A. Popowicz, J.M. Manning, and E.J. Neufeld Hemoglobin Jamaica Plain--A Dominant Sickling Hemoglobin with Reduced Oxygen Affinity. New England Journal of Medicine, 315,
40-46 (2004).

Y. Zhang, L.R. Manning, J.Falcone, O.Platt, and J.M. Manning. Human Erythrocyte Membrane Band 3 Protein Influences Hemoglobin Cooperativity. Possible Effect on Oxygen Transport. J.Biol.Chem.  278,39565-39571 (2003).

X. Li, R.W. Briehl, R.M. Bookchin, R. Josephs, B. Wei, J.M. Manning, and F.A. Ferrone. Sickle Hemoglobin Polymer Stability Probed by Triple and Quadruple Mutant Hybrids. J. Biol. Chem. 277, 13479-13487 (2002).

T. Yagami, B.T. Ballard, J.C. Padovan, B.T. Chait, A.M. Popowicz, and J.M. Manning. N-Terminal Contributions of the y-Subunit of Fetal Hemoglobin to its Tetramer Strength Remote Effects of Subunit Contacts. Protein Science. 11, 27-35 (2002).

K. Kishimoto and J.M. Manning. Adherence of Vancomycin to Proteins. J. Protein Chem. 20, 455-461 (2001).

L.R. Manning and J.M. Manning. The Acetylation State of Human Fetal Hemoglobin Modulates the Strength of Its Subunits Interactions: Long-Range Effects and Implications for Histone Interactions in the Nucleosome. Biochemistry. 40, 1635-1639 (2001).

K. Inagaki, J. Inagaki, A. Dumoulin, J.C. Padovan, B.T. Chait, A. Popowicz, L.R. Manning, and J.M. Manning. Expression and Properties of Recombinant HbA2(a2d2) and Hybrids Containing d-b Sequences. J. Prot. Chem. 19, 647-660 (2000).

P.W. van Ophem, B.W. Lepore, K. Kishimoto, D. Ring, and J.M. Manning. Studies on an Active Site Mutant, E177, That Affects Binding of the Coenzyme on a Suicide Substrate. Biochemistry and Molecular Biology of Vitamin B6 and PQQ-Dependent Proteins. (Eds. A. Irairte, H.M. Kagan, and M. Martinez-Carrion). 339-349 (2000).

Weihua Chen, A. Dumoulin, X. Li, J.C. Padovan, B.T. Chait, R. Buonopane, O.S. Platt, L.R. Manning, and J.M. Manning. Transposing Sequences between Fetal anad Adult Hemoglobins Indicates Which Subunits and Regulatory Molecule Interfaces are Functionally Related. Biochemistry. 39, 3774-3781 (2000).

D.A. Bulik, P. van Ophem, J.M. Manning, D.S. Newburg, and E.L. Jarroll. UDP-N-Acetylglucosamine Pyrophosphorylase, A Key Enzyme in Encysting Giardia, is Allosterically Regulated. J. Biol. Chem. 275, 14722-14728 (2000).

Kazuhiza Kishimoto, C. Yasuda, and J.M. Manning. Reversible Disssociation/Association of D-Amino Acid Transaminase Subunits: Properties of Isolated Dimers and Inactive Monomers. Biochemistry. 39, 381-387 (2000).

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