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Preparation and Analysis of Protein Crystals

In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.

Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes.

Fungal cellulases offer advantages of a secreted enzyme complex and relative easiness and economy of producing enzyme. Considerable amount of work has been done on fungal cellulases, especially with resurgence of interest in biomass-ethanol and concept ofbio-refineries. Significant information has also been gained on basic biology of organisms producing cellulases, and in process development for enzyme production and biomass saccharification. This review addresses developments in the field of fungal cellulases for lignocellulose degradation.

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Progress in research on fungal cellulases for lignocellulose degradation

The invention provides reagents, kits and methods for detecting and/or quantifying proteins in complex mixtures, such as a cell lysate. The methods can be used in high throughput assays to profile cellular proteomes. In one aspect, the invention provides a peptide internal standard labeled with a stable isotope and corresponding in amino acid sequence to the amino acid sequence of a subsequence of a target polypeptide. In another aspect, the peptide internal standard is labeled at a modified amino acid residue and is used to determine the presence of, and/or quantitate the amound of a particular modified form of a protein.

Absolute quantification of proteins and modified forms thereof by multistage mass spectrometry

Thermophilic fungal cellulases are promising enzymes in protein engineering efforts aimed at optimizing industrial processes, such as biomass degradation and biofuel production. The cloning and expression in recent years of new cellulase genes from thermophilic fungi have led to a better understanding of cellulose degradation in these species. Moreover, crystal structures of thermophilic fungal cellulases are now available, providing insights into their function and stability. The present paper is focused on recent progress in cloning, expression, regulation, and structure of thermophilic fungal cellulases and the current research efforts to improve their properties for better use in biotechnological applications.

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Cellulases from thermophilic fungi: recent insights and biotechnological potential.

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have extended our previous work on the structural and biochemical diversity of GH 12 homologs to include the most stable fungal GH 12 found, Humicola grisea Cel12A. The H. grisea enzyme was much more stable to irreversible thermal denaturation than the Trichoderma reesei enzyme. It had an apparent denaturation midpoint (Tm) of 68.7°C, 14.3°C higher than the T. reesei enzyme. There are an additional three cysteines found in the H. grisea Cel12A enzyme. To determine their importance for thermal stability, we constructed three H. grisea Cel12A single mutants in which these cysteines were exchanged with the corresponding residues in the T. reesei enzyme. We also introduced these cysteine residues into the T. reesei enzyme. The thermal stability of these variants was determined. Substitutions at any of the three positions affected stability, with the largest effect seen in H. grisea C206P, which has a Tm 9.1°C lower than that of the wild type. The T. reesei cysteine variant that gave the largest increase in stability, with a Tm 3.9°C higher than wild type, was the P201C mutation, the converse of the destabilizing C206P mutation in H. grisea. To help rationalize the results, we have determined the crystal structure of the H. grisea enzyme and of the most stable T. reesei cysteine variant, P201C. The three cysteines in H. grisea Cel12A play an important role in the thermal stability of this protein, although they are not involved in a disulfide bond.

The Humicola grisea Cel12A enzyme structure at 1.2 A resolution and the impact of its free cysteine residues on thermal stability.

The present invention makes use of unique tags of a specific biopolymer that can be exploited for determining the concentration the biopolymer in crude solutions. In preferred embodiments the biopolymer is either a protein or a polynucleotide. Particularly, the invention provides a method for the determination and quantitation of biomolecules in crude mixtures by way of a separatioln technique in combination with mass spectroscopy. In one general embodiment, a target biomolecule is selected for analysis and an analog thereof is generated. Peak area integration of the peptide pairs provides a direct measure for the amount of target protein in the crude solution.

Sigrid Paech

Papers

The Humicola grisea Cel12A enzyme structure at 1.2 A resolution and the impact of its free cysteine residues on thermal stability.

Mass spectrometric analysis of biopolymers