CRY2 Variant Photosensitivity Mutation for Low Light Dimerization
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Solution Overview
Problem
Current methods for inducing protein dimerization, such as chemically induced homo-association, have limitations in spatiotemporal resolution and diversity, making it difficult to study dynamic protein activity, and wild-type CRY2 protein dimerization is only possible under high light irradiation conditions.
Innovation Solution
A CRY2 variant is developed with increased photosensitivity by substituting specific amino acids in the N277SEGE281 sequence, allowing dimerization under low light illumination, and a Ca2+ modulator is created by fusing the CRY2 variant with STIM1 protein to control Ca2+ release through light irradiation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If wild-type CRY2 protein is used for dimerization, then dimerization can occur, but only under high light irradiation conditions
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acids (E279A, E281A, G280W) in the CRY2 protein sequence to alter its photosensitivity parameters. These mutations change the protein's light absorption characteristics and dimerization threshold, enabling dimerization to occur under low light irradiation conditions instead of requiring high intensity light, thus resolving the contradiction between illumination intensity requirements and ease of operation
2Reliability
If chemically induced homo-association is used to induce protein dimerization, then dimerization can be achieved, but spatiotemporal resolution is low and diversity is limited
Solution Approach 1:
The patent replaces chemical induction methods with optical induction using light-activated CRY2 protein. This substitution enables precise spatiotemporal control of dimerization through light delivery, allowing researchers to induce dimerization at specific locations and times with high precision, while maintaining reliable dimerization through the photostimulated conformational change of CRY2
Solution Approach 2:
The patent introduces dynamic control capability through light-activated CRY2, which can be switched between monomeric and dimeric states on-demand via light irradiation. This dynamic system allows real-time manipulation of protein dimerization in response to temporal and spatial light patterns, achieving both high measurement precision and reliable dimerization control
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The CRY2 variant enables effective regulation of cellular signals even under low light conditions, and the Ca2+ modulator allows for non-invasive light-induced Ca2+ introduction in cells, enhancing the precision of cellular signal transmission and behavioral studies.
Implementation Method 1
CRY2 variants according to embodiments are CRY2 variants in which at least one of E279 and E281 having a negative charge among a N277SEGE281 sequence of SEQ ID NO: 1 is substituted with any one selected from the neutral amino acid group of alanine (A), isoleucine (I), leucine (L), valine (V), phenylalanine (F), methionine (M), and tryptophan (W), or S278 or G280 in the N277SEGE281 sequence of SEQ ID NO: 1 is substituted with any one selected from a bulky amino acid group of tryptophan (W) and phenylalanine (F). The CRY2 variant according to embodiments may be dimerized even under low light illumination.
Implementation Method 2
The Ca2+ modulator according to embodiments includes a CRY2 variant and a STIM1 protein bound to the N-terminus or C-terminus of the CRY2 variant. The Ca2+ modulator allows for non-invasive light-induced Ca2+ introduction in cells.
Data Source
AI summary
Provided is a CRY2 variant in which E279 and/or E281 having a negative charge in a N277SEGE281 sequence of SEQ ID NO: 1 is substituted with any one selected from the neutral amino acid group of alanine (A), isoleucine (I), leucine (L), valine (V), phenylalanine (F), methionine (M) and tryptophan (W), or in which S278 or G280 of a N277SEGE281 sequence of SEQ ID NO: 1 is substituted with any one selected from the bulky amino acid group of tryptophan (W) and phenylalanine (F).