Structural Characterization of Proteins

Structural proteomics aims to achieve a structural characterization of protein complexes. Structural genomics is sometimes used to describe the goal of delineating the 3D structures for individual proteins.

There are a number of experimental and theorectical methods used in structural proteomics:

Experimental Methods:

1) X-ray crystallography: is the most prolific technique for the structural analysis of proteins and protein complexes. Crystallography requires that milligram quantities of a pure protein can be prepared, and that the protein can be induced to form 3D crystals. When suitable crystals and high resolution cystallographic data are obtained, there is little need for other methods of structure characterization.

2) NMR spectroscopy: is also significant in determining the number of structures in the database of biomelecular structures. Although NMR analysis is generally not as applicable as X-ray crystallography to protein structures with more than 300 amino acid residues, it is more suitable than x-ray crystallography to study their dynamics and interactions in solution.

3) X-ray crystallography or two-dimensional electron microscopy (2D EM): produces images that represent only 2D projections of the specimen. Nevertheless, the full 3D structure of the object can be reconstructed again if one is able to start with many such projections, each showing the object from a different angle. Because it is possible to use non-crystalline particles, the purity does not need to be at that standard required for crystallization.

4) Electron tomography: is based upon multiple tilted views of the same object. Tomograms of cells are essentially 3D images of the cell's entire proteome. They reveal information about the spatial relationships of macromolecules in the cytoplasm.

5) Immuno-electron microscopy: uses a construct of the protein of interest that binds to a gold-labelled antibody. The relative position of the gold particles is then identified by EM.

6) Chemical crosslinking with mass spectroscopy: relies on crosslinking reagents that covalently link proteins interacting with each other. Proteolytic digestion and subsequent mass spectroscopic identification of the crosslinked species reveal their composition.

7) Affinity purification with mass spectroscopy: combines purification of protein complexes with identification of their individual components by mass spectroscopy.

8) Fluorescence resonance energy transfer (FRET):

9) site-directed mutagenesis: can reveal which subunits in a complex interact with each other.

10) Yeast two-hydrid system:

11) Protein arrays:

Non Experimental Methods:

1) de novo or ab initio methods: predicts the structure from sequence alone, without relying on similarity at the fold level between the modeled sequence and any known structures. These methods assume that the native structure corresponds to the global free-energy minimum and attempts to find this minimum by an exploration of many conceivable protein conformations.

2) homology modeling: relies on detectable similarity spanning most of the modeled sequence and at least one known structure. It relies on finding known structures related to the sequence to be modeled, aligning the sequence with the related structures, building a model, and assessing the model.

3) computational docking: is based on maximizing the shape and chemical complementarities between a given pair of interacting proteins.

4) bioinformatic analysis of genomic sequences: Multiple sequence alignments and protein structures may indicate the presence and location of protein interaction interfaces using this approach.

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