Enhanced fluorescent proteins can be widely used to track the localization and dynamics of proteins, organelles, and other cellular compartments, as well as a tracer of intracellular protein trafficking. However, mutagenesis has provoked an assortment of visible fluorescent proteins, and besides mutations that influence spectral properties, numerous modifications have been described that enhance the brightness of one or more visible fluorescent protein variants.

In this contribution, the effects of a particular mutation in the yellow fluorescent protein (YFP) enhanced variant, Venus, are described according to what was reported by Kremers et al. (2006). Venus is an essential (constitutively fluorescent) yellow fluorescent protein firstly characterized in 2002, derived from Aequorea victoria (Nagai et al., 2002). Among its characteristics, Venus contains a novel mutation, F46L, folding relatively well and acquiring tolerance to exposure to acidosis and chloride ions, compared to other yellow fluorescent proteins.

Nevertheless, Kremers et al. (2006) report the results of a site-directed mutagenesis approach to improve Venus's thermosensitivity, and dim fluorescence through the folding mutation A206K, named as mVenus. According to Zacharias et al. (2002), A206K has been described to abolish the tendency of YFP to dimerize. To compare a variety of mutations, Kremers et al. (2006) measured the time course of fluorescence development in Escherichia coli cultures expressing Venus’s variants as GST-tagged fusion proteins at 37 °C. The growth rate of all cultures was similar; therefore, no correction for bacterial growth was required. Results obtained by Kremers et al. (2006) are shown in the following image:

According to what is stated in

Figure 1

, Kremers et al. (2006) state that A206K greatly improved protein folding and solubility and enabled more protein to become fluorescent compared to the original Venus. Moreover, A206K variant or

mVenus

, reduces the aggregation of yellow fluorescent GST-fusion proteins. For instance, we recommend using mVenus variant for following fluorescent applications, such as dual imaging or fluorescence resonance energy transfer (FRET) sensors. Next, we present the three-dimensional structure of mVenus generated by AlphaFold2 using MMSeqs2 (Mirdita et al., 2022). This structure is as follows: