Main Research Interests

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My current research interests derive from my background in theoretical biophysics and in experimental X-ray crystallography, and aim at developing new methodologies for structural biophysics. In particular, I am interested in extending the structural information gained from classical X-ray crystallography experiments, either by combining it with other methods such as Electron Microscopy, or by improving the X-ray experiment itself.

Below are listed a few projects on which I have worked over the last few years.

(1) Structural studies by X-ray crystallography

The first project described here was the main focus of my thesis work.

(1.1) Amidation of bioactive peptides

(with Mario Amzel, Sean Prigge, Ninian Blackburn, Betty Eipper and Richard Mains)

Amidation of the COOH-terminus of bioactive peptides (e.g. neuropeptides, hormones peptides) is essential for their biological activity. This posttranslational modification requires the sequential action of a monooxygenase (PHM = peptidylglycine alpha-hydroxylating monooxygenase) and a lyase (PAL = peptidyl-alpha-hydroxylglycine alpha-amidating lyase). The structures of PHM [1] and of PHM complexes [2,3] were recently determined by our group and provide a framework for understanding the chemical and electron-transfer steps involved in catalysis.

This project involves structural studies of PHM mutants designed to probe the catalytic mechanism [4]. These structural studies are complemented by quantum-chemical calculations.

We are also undertaking the expression, purification and structure determination of PAL, the second domain of PAM, to provide a complete description of the peptide amidation reaction.

References:

  1. Siebert X., Eipper B.A., Mains R.E., Prigge S.T., Blackburn N.J. and Amzel L.M. ``The catalytic copper of Peptidylglycine alpha-Hydroxylating Monooxygenase also plays a critical structural role.'', Biophysical Journal (accepted July 2005).
  2. Prigge S.T., Eipper B.A., Mains R.E., Amzel L.M. "Dioxygen binds end-on to mononuclear copper in a precatalytic enzyme complex.", Science (2004), 304(5672):864-7.
  3. Prigge S.T., Kolhekar A.S., Eipper B.A., Mains R.E., Amzel L.M., "Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase.", Nat. Struct. Biol. (1999), 6:976-983
  4. Prigge S.T., Kolhekar A.S., Eipper B.A., Mains R.E., Amzel L.M. "Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase.", Science (1997), 278:1300-1305

(1.2) Amyloidogenic proteins (older stuff)

The main features of Alzheimer's disease are extracellular plaques formed by fibrillar aggregate of normally soluble proteins and neurofibrillary tangles. The main constituent of these plaques is a sticky protein called beta-amyloid, which also associates with other proteins from the cerebrospinal fluid (i.e. transthyretin, antichymotrypsin, ...). The purpose of this project is to study complexes of these proteins with the beta-amyloid peptide to determine their role in amyloid plaques formation.

(2) Statistical mechanics of macromolecular association

Molecular association is opposed by a loss of entropy due to the restriction of motion of each of the components in the complex. This entropy loss is difficult to estimate from first principles, because of the complexity of the intermolecular interactions in solution. We have recently proposed a statistical-mechanical framework, based on cell theories of condensed phases, to study the different components (e.g. translational, rotational) of this entropy loss. This framework has been tested using molecular dynamics simulations on simple systems (pure water, small molecules in water), as well as macromolecular assemblies (benzene binding to lysozyme). The translational entropy of a solute in water appears to be correlated with the size and the polarity of this solute.

References:

  1. Siebert X. and Amzel L. M., Loss of Translational Entropy in Molecular Associations Proteins 54:104--115 (2004) .
  2. You can also get a preprint of this work [here] .

(3) Hydrophobic effects in ligand binding

This work has been done in collaboration with Gerhard Hummer at Los Alamos National Laboratories, and at the National Institute of Health.

The goal of this project is to map the hydrophobic character of protein surfaces. Hydrophobic maps are a powerful tool to identify potential ligand binding sites, or to optimize existing ligands. We use a three-fold approach, combining extensive molecular dynamics simulations of proteins in solution, information theory calculations [1], and a new hydrophobic force-field [2]. We have applied this approach to inhibitor binding to the HIV-1 gp41 fusion peptide. This study shows good agreement between experimental and theoretical results, and provides guidance for the design of optimized inhibitors [3].

References:

  1. Hummer G, Garde S, Garcia AE, Pohorille A, Pratt LR. An information theory model of hydrophobic interactions. Proc. Nat. Acad. Sci. USA (1996) 93:8951-8955
  2. Hummer G. Hydrophobic force field as a molecular alternative to surface-area models. J. Am. Chem. Soc. (1999) 121:6299-6305
  3. Siebert X. and Hummer G. Hydrophobicity Maps of the N-Peptide Coiled Coil of HIV-1 gp41 , Biochemistry (2002), 41 (9), 2956--2961.