Glycan-specific analytical tools

1. A method for generating a lectenz comprising an inactivated mutated carbohydrate-processing enzyme having enhanced affinity for its substrate compared to a corresponding wild-type carbohydrate-processing enzyme, the method comprising:

(a) providing a 3D structure of an enzyme-substrate complex comprising a substrate bound to a catalytically inactive mutant carbohydrate-processing enzyme or a corresponding wild-type carbohydrate-processing enzyme, wherein the catalytically inactive carbohydrate-processing enzyme comprises an amino acid sequence comprising at least one inactivating mutation that eliminates catalytic activity of the enzyme;

(b) performing a computational simulation on the enzyme-substrate complex to predict the per-residue contributions to total interaction energy (ΔE MM ) and/or total binding free energy (ΔG Binding ) for amino acid residues of the enzyme;

(c) analyzing the per-residue energetic contributions to identify at least one amino acid residue as a potential mutation site for enhancing binding affinity of the inactivated enzyme for its substrate as compared to a wild-type enzyme, wherein an amino acid residue is identified as a potential mutation site for enhancing binding activity when

(i) for an amino acid residue located within 5 Å of the substrate in the enzyme-substrate complex, the per-residue contribution of the amino acid residue to at least one of ΔE MM or ΔG Binding is ≥−0.7 kcal/mol; and

(ii) for an amino acid residue located more than 5 Å from the substrate in the enzyme-substrate complex, the per-residue contribution of the amino acid residue to at least one of ΔE MM or ΔG Binding is ≥0.0 kcal/mol;

(d) expressing a library of inactivated mutated carbohydrate-processing enzymes, each inactivated mutated enzyme comprising a plurality of amino acid mutations, wherein

each inactivated mutated enzyme comprises at least one inactivating mutation that eliminates catalytic activity of the enzyme; and

each inactivated mutated enzyme further independently comprises at least one potential affinity-enhancing mutation at a site identified in step (c);

(e) assaying the inactivated mutated enzymes of step (d) for their ability to form the enzyme-substrate complex; and

(f) identifying inactivated mutated enzymes from step (e) that exhibit binding affinities to the substrate that are at least 1.2-fold greater than those of the wild-type carbohydrate-processing enzyme.

2. The method of claim 1 , wherein at least one potential affinity-enhancing mutation is an amino acid substitution that replaces an amino acid residue located within 5 Å of the substrate when bound to the wild-type or inactivated enzyme.

3. The method of claim 2 , wherein at least one amino acid being replaced makes a favorable (<0.0 kcal/mol) per-residue contribution to the total interaction energy (ΔE MM ) or total binding free energy (ΔG Binding ).

4. The method of claim 3 , wherein the per-residue contribution of the amino acid being replaced is between −0.5 kcal/mol and 0.0 kcal/mol.

5. The method of claim 1 , wherein at least one potential affinity-enhancing mutation is an amino acid substitution that replaces an amino acid that makes an unfavorable (>0.0 kcal/mol) per-residue contribution to the total interaction energy (ΔE MM ) or total binding free energy (ΔG Binding ).

6. The method of claim 1 , wherein at least one of step (b) or step (c) comprises computational alanine scanning mutagenesis, direct ΔG calculations, or thermodynamic integration calculations.

7. The method of claim 1 , wherein the simulation comprises molecular dynamics simulation, Monte Carlo simulation, or side-chain rotamer searching.

8. The method of claim 1 , wherein the simulation is performed using classical force fields.

9. The method of claim 1 , wherein the library of step (d) comprises inactivated mutated enzymes comprising the same inactivating mutation.

10. The method of claim 1 , wherein the library of step (d) comprises inactivated mutated enzymes comprising different inactivating mutations.

11. The method of claim 1 , wherein the library is a randomized library.

12. The method of claim 1 , wherein the 3D structure of the enzyme-substrate complex is an X-ray structure.

13. The method of claim 12 , wherein the X-ray structure represents an inactive enzyme-substrate complex, an active enzyme-inhibitor complex, or an enzyme-product complex.

14. The method of claim 1 , further comprising generating the 3D structure using homology or comparative modeling.

15. The method of claim 1 , further comprising generating the 3D structure from crystallographic data from a free enzyme by predicting a co-complex with a substrate using a computational docking program.

16. The method of claim 1 , wherein the carbohydrate-processing enzyme is PNGaseF, and wherein the inactivated mutated carbohydrate-processing enzymes of step (d) comprise amino acid mutations at D57, Y62, E118, S155, 1156, G192 and E206.

17. A method for generating a novel biomolecule, wherein the biomolecule comprises a carbohydrate-binding protein having one or more mutations that:

(i) increase the affinity of the protein to its carbohydrate ligand, as compared to a wild-type protein; and

(ii) have substantially no alteration in the specificity of the protein;

the method comprising:

(a) performing a computational simulation on the carbohydrate-protein complex to predict per-residue contributions to total interaction energy (ΔE MM ) or total binding free energy (ΔG Binding ) for amino acid residues of the protein;

(b) analyzing the per-residue energetic contributions to identify at least one amino acid residue as a potential mutation site for enhancing binding affinity of the protein for the carbohydrate ligand as compared to a wild-type protein, wherein an amino acid residue is identified as a potential mutation site for enhancing binding affinity when the amino acid residue is located within 5 Å of the carbohydrate ligand in the carbohydrate-protein complex, and the per-residue contribution of the amino acid residue to at least one of ΔE MM or ΔG Binding is ≥−0.7 kcal/mol;

(c) expressing a library of mutant carbohydrate-binding proteins, wherein each mutant carbohydrate-binding protein independently comprises at least one potential affinity-enhancing amino acid substitution that replaces an amino acid residue identified in step (b);

(d) assaying the mutant proteins of step (c) for their ability to form the carbohydrate-protein complex and for binding specificity; and

(e) identifying mutant proteins from step (d) that exhibit binding affinities to the carbohydrate that are at least 1.2-fold greater than those of wild-type protein.

18. The method of claim 17 , wherein the biomolecule is a lectin or an antibody.