The Structure Determination of M. denitrificans Cytochrome C₅₅₀

The protein sequence and the three-dimensional folding structure for cytochrome c550 from Micrococcus denitrificans are reported. The tertiary structure resulted from an x-ray crystallographic determination. Standard protein chemistry techniques were applied to sequence all the fragment peptides from a tryptic digest of c550. This information was combined with a 2.45 Å electron density map of the protein where identifying the tryptic fragments in the map led to the correct ordering of the peptides and the final complete sequence. The crystallization of c550 is described. Three chemical derivatives of the protein were found, PtCl42-; UO22+, and Pt(CN)42-, and these aided in the isomorphous replacement solution to the crystallographic phase problem. The interpretation and refinement of the derivatives to produce an accurate protein map is discussed. This bacterial c550 possesses a structure similar to the known cytochromes from eukaryotic sources and from a photosynthetic bacterium. The three are contrasted to conclude that for the cytochrome family, there exists a core structure of polypeptide folding. Current proposed mechanisms for the action of c-type cytochromes are rejected as being incompatible with the c550 structure. The view is advocated that the exposed heme edge in cytochrome may be the sole active site for the molecule.</p

Automatic rebuilding and optimization of crystallographic structures in the Protein Data Bank

Motivation: Macromolecular crystal structures in the Protein Data Bank (PDB) are a key source of structural insight into biological processes. These structures, some >30 years old, were constructed with methods of their era. With PDB_REDO, we aim to automatically optimize these structures to better fit their corresponding experimental data, passing the benefits of new methods in crystallography on to a wide base of non-crystallographer structure users

Amination of enzymes to improve biocatalyst performance: coupling genetic modification and physicochemical tools

Improvement of the features of an enzyme is in many instances a pre-requisite for the industrial implementation of these exceedingly interesting biocatalysts. To reach this goal, the researcher may utilize different tools. For example, amination of the enzyme surface produces an alteration of the isoelectric point of the protein along with its chemical reactivity (primary amino groups are the most widely used to obtain the reaction of the enzyme with surfaces, chemical modifiers, etc.) and even its “in vivo” behavior. This review will show some examples of chemical (mainly modifying the carboxylic groups using the carbodiimide route), physical (using polycationic polymers like polyethyleneimine) and genetic amination of the enzyme surface. Special emphasis will be put on cases where the amination is performed to improve subsequent protein modifications. Thus, amination has been used to increase the intensity of the enzyme/support multipoint covalent attachment, to improve the interaction with cation exchanger supports or polymers, or to promote the formation of crosslinkings (both intra-molecular and in the production of crosslinked enzyme aggregates). In other cases, amination has been used to directly modulate the enzyme properties (both in immobilized or free form). Amination of the enzyme surface may also pursue other goals not related to biocatalysis. For example, it has been used to improve the raising of antibodies against different compounds (both increasing the number of haptamers per enzyme and the immunogenicity of the composite) or the ability to penetrate cell membranes. Thus, amination may be a very powerful tool to improve the use of enzymes and proteins in many different areas and a great expansion of its usage may be expected in the near future.This work has been supported by grant CTQ2013-41507-R from Spanish MINECO, grant no. 1102-489-25428 from COLCIENCIAS and Universidad Industrial de Santander (VIE-UIS Research Program) and CNPq and FAPERGS (Brazil). A. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovacion for a Ramon y Cajal fellowship (RyC-2009–03813)