Table 2. DNA binding, NAD⁺ binding and catalytic activity of ERM-BP-WT and ERM-BP mutants.

Wild type (WT) and mutant ERM-BP proteins were analyzed by three approaches. DNA binding was determined by EMSA analysis with increasing concentration of NAD⁺. Protein thermal shift assay was performed to determine NAD⁺ binding and melting temperature (Tm) in the presence of varying concentration of NAD⁺ is shown. Nicotinamidase activity was determined by measuring conversion of nicotinamide to nicotinic acid by HPLC.

Proteins	DNA Binding (EMSA) NAD+ (mM)#			NAD+ Binding (Tm) NAD+ (mM)§				Nicotinamidase activity (% conversion)¶
	0	1	4	0	1	4	P-values (0-4)
1. GST†	-	-	-	52 ± 7.0	53 ± 7.0	51 ± 7.0	0.6549	no conversion
2. ERM-BP-WT‡	++	+++	++++	57 ± 0.3	60 ± 0.7	62 ± 1.3	1.1E-08	52 ± 0.6
3. D12A‡	+/-	++	+++	57 ± 1.0	59 ± 1.0	62 ± 1.0	1.5E-08	no conversion
4. ERM-BP-DBM‡	-	-	++	57 ± 0.3	58 ± 0.8	61 ± 1.0	9.9E-09	50 ± 2.1
5. K150A‡	+/-	++	+++	58 ± 1.7	59 ± 1.0	62 ± 11	3.7E-05	no conversion
6. C198A†	-	-	-/+	57 ± 0.3	57 ± 0.4	57 ± 0.5	0.3796	no conversion

# EMSA (Electrophoretic mobility shift assay) with varying amounts of NAD⁺ (0, 1, 4 mM): ‘++++' indicates strong binding, ‘+++' and ‘++' indicate moderate, ‘+/-' indicates weak binding and '-'“indicates no binding. § NAD⁺ binding was monitored by protein thermal shift assay with varying amounts of NAD⁺ (0, 1, 4 mM). The Tm (melting temperature) is shown as mean ±s.d. (n = 3) Student’s t-test; p-values are shown between NAD⁺ 0 mM and 4 mM concentrations. ¶ The turnover of nicotinamide to nicotinic acid by ERM-BP-WT and mutant recombinant proteins are shown as percentage conversion mean ±s.d. (n = 3). ‡ Indicates having significant effects in DNA /NAD⁺ binding and catalytic activity, † Indicates not having significant effects in DNA/NAD⁺ binding and catalytic activity.