FP was measured with a PerkinElmer EnVision multi-label plate reader (485 nm excitation and 520 nm emission). Three 25-μl aliquots of each reaction mixture were transferred to a black opaque 384-well plate and incubated for 30 min at room temperature in the dark before measurement. The final protein concentrations were 1.5, 3, 5, 10, 20, 30, 50, 75, 100, 150, 300, and 600 n m, and the final probe concentration was 5 n m. For binding reactions, 20 μl of 25 n m labeled probe was added to 80 μl of protein in FP buffer. The 16 mutant proteins were separated into four independent sets of experiments, each including the wild-type protein as a control. All FP assays were conducted in FP buffer. The protein concentration was quantified using A 280 (NanoDrop), and purity was confirmed by SDS-PAGE. The FAM-labeled dsDNA probe was then diluted to 25 n m with FP buffer (30 m m HEPES, 50 m m potassium chloride, 10 m m guanidinium chloride, 2 m m magnesium chloride, 0.5 m m EDTA, and 0.01% Nonidet P-40 (pH 7.5)). An unlabeled probe for competition was made in a similar way using JM569 (5′-AGT CAG TCA GTC AGT-3′) and JM570 (5′-ACT GAC TGA CTG ACT-3′) diluted to 50 μ m in water. The dsDNA probes were made by combining JM565 (FAM-AGT CAG TCA GTC AGT-3′) with JM566 (FAM-ACT GAC TGA CTG ACT-3′) to a final concentration of 0.5 μ m in water, placing them in boiling water for 5 min, and allowing the solution to cool at room temperature. The aforementioned mutants and wild-type VRN1(208–341) were assayed for in vitro DNA-binding activity using a fluorescence polarization (FP) assay that measured fluorescence upon binding of each protein to 6-carboxyfluorescein (FAM)-labeled dsDNA probes. All proteins were 15N-labeled, and an HSQC spectrum was acquired for each as previously described ( A final set of four mutants were made by incorporating R249E as a single, second, or third mutation with the appropriate template using primer JM577 (5′-AGA GTG GTT CTG GAG CCA TCC TAT CTA-3′) and its complement. The PCR product was ligated to create the mutant referred to hereafter as loopΔ. The bases encoding ISEKSSKS are shown in lowercase letters and underlined. To replace the loop of VRN1 with the sequence RPSYLYRGCI with the equivalent At1g16640-derived loop sequence ISEKSSKS, PCR was done using phosphorylated primers JM563 (5′- tct agt aaa tcc ATG TAT CTT CCT TCT GGG-3′) and JM564 (5′- ttt ctc aga gat CAG AAC CAC TCT GAA GAA-3′). To make the R289A/R296A double mutant, the aforementioned R289A plasmid was mutagenized by PCR with a shorter primer for R296A (5′-TAC AAA GCC GGG GCG GCC AAA TTC AGT-3′). To make the R289E/R296E double mutant, the single mutations were introduced sequentially. Subsequent to these six mutants, another group of six mutants were made with less conservative changes, including G295E (5′-CTC TAC AAA GCC GAG AGA GCC AAA TTC-3′), R289E (5′-CAA TGG CCT GTT GAG TGT CTC TAC AAA-3′), and R296E (5′-TAC AAA GCC GGG GAG GCC AAA TTC AGT-3′. A two-dimensional 1H- 15N HSQC spectrum was acquired for each mutant to ascertain the effect of each mutation on the protein. TABLE 1 Data collection, phasing, and refinement statisticsįigure of merit (before/after density modification) Thus, we have revealed that although VRN1 is sequence-nonspecific in DNA binding, it has a defined DNA-binding surface. The triple mutant R249E/R289E/R296E was almost completely incapable of DNA binding in vitro. We established the DNA-binding face using NMR and then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions, ensuring that the protein fold was not disturbed using heteronuclear single quantum correlation NMR spectra. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs but absent in other B3 domains. To understand its sequence-nonspecific binding, we crystallized VRN1(208–341) and solved its crystal structure to 1.6 Å resolution using selenium/single-wavelength anomalous diffraction methods. In this work, we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and shows sequence-nonspecific binding in vitro. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana, and one of these is critical for accelerating flowering in response to prolonged cold treatment, an epigenetic process called vernalization. The B3 DNA-binding domain is a plant-specific domain found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Glycobiology and Extracellular Matrices.
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