# US 10,373,702 B2 · Water-soluble trans-membrane proteins and methods for the preparation and use thereof

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- Number: US 10,373,702 B2
- Document ID: US10373702B2
- Status: Granted
- Kind: B2
- Application number: 14/723,399
- Filed: 2015-05-27
- Published: 2015-12-24
- Granted: 2019-08-06
- Earliest claimed: 2014-03-27
- Assignees: Massachusetts Institute of Technology
- Applicants: Massachusetts Institute of Technology
- Inventors: Shuguang Zhang; Fei Tao
- Web page: https://patentgrep.com/patent/US10373702B2

## CPC

- G16B 15/20: Protein or domain folding
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/723: {G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor}
- G16B 15/00: ICT specially adapted for analysing two-dimensional [2D] or three-dimensional [3D] molecular structures, e.g. structural or functional relations or structure alignment
- G16B 35/00: ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
- G16B 35/10: Design of libraries
- G16C 20/60: In silico combinatorial chemistry
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G16C: COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE

## Abstract

The present invention is directed to a computer implemented method for executing a procedure to select a water-soluble variant of a G Protein-Coupled Receptor (GPCR).

## Claims

### Claim 1

A method for producing a water-soluble variant of a G Protein-Coupled Receptor (GPCR), the method comprising:
(1) entering a sequence of the GPCR for analysis;
(2) obtaining a variant of the GPCR, wherein substantially all of Leucine (L), Isoleucine (I), Valine (V), and Phenylalanine (F) in the transmembrane (TM) domain alpha-helical segments (“TM regions”) of the GPCR are substituted, wherein:
(a)
each said Leucine (L) is independently substituted by Glutamine (Q), Asparagine (N), or Serine (S);
(b) each said Isoleucine (I) and said Valine (V) are independently substituted by Threonine (T), Asparagine (N), or Serine (S); and,
(c) each said Phenylalanine is substituted by Tyrosine (Y); and, subsequently,
(3) obtaining an α-helical secondary structure result for the variant to verify maintenance of α-helical secondary structures in the variant;
(4) obtaining a trans-membrane (TM) region result for the variant to verify water solubility of the variant, wherein said water solubility of said variant is verified when said TM region result predicts absence of TM regions or low propensity to form TM regions, based on the sequence of said variant, and,
(5) selecting and producing the variant obtained in step (2) that has maintained α-helical secondary structures according to step (3), and is verified to have water solubility in step (4) as said water-soluble variant of the GPCR.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (3) is performed prior to, concurrently with, or after step (4).

### Claim 3 (depends on 1)

The method of claim 1 further comprising performing the method with a data processor.

### Claim 4 (depends on 1)

The method of claim 1, wherein about 96%, 97%, 98%, 99% or 100% of said leucines are substituted by glutamines, about 96%, 97%, 98%, 99% or 100% of said isoleucines are substituted by threonines, about 96%, 97%, 98%, 99% or 100% of said valines are substituted by threonines, or about 96%, 97%, 98%, 99% or 100% of said phenylalanines are substituted by tyrosines.

### Claim 5 (depends on 1)

The method of claim 1, wherein 1, 2, or 3 of said leucines are not substituted, 1, 2, or 3 of said isoleucines are not substituted, 1, 2, or 3 of said valines are not substituted, or 1, 2, or 3 of said phenylalanines are not substituted.

### Claim 6 (depends on 1)

The method of claim 1, wherein the TM regions of the GPCR are predicted using TMHMM 2.0 (TransMembrane prediction using Hidden Markov Models) software module.

### Claim 7 (depends on 1)

A non-transitory computer readable medium having stored thereon a sequence of instructions to perform the method of claim 1.

### Claim 8 (depends on 1)

The method of claim 1, wherein in step (2), all (100% of) said leucines in the TM regions are substituted by glutamines, all (100% of) said isoleucines in the TM regions are substituted by threonines, all (100% of) said valines in the TM regions are substituted by threonines, and all (100% of) said phenylalanines in the TM regions are substituted by tyrosines.

### Claim 9 (depends on 1)

The method of claim 1, wherein all said leucines are substituted by glutamines, all said isoleucines are substituted by threonines, all said valines are substituted by threonines, or all said phenylalanines are substituted by tyrosines.