Dynamics of a family of cyan fluorescent proteins probed by incoherent neutron scattering

Abstract

<jats:p>Cyan fluorescent proteins (CFPs) are variants of green fluorescent proteins in which the central tyrosine of the chromophore has been replaced by a tryptophan. The increased bulk of the chromophore within a compact protein and the change in the positioning of atoms capable of hydrogen bonding have made it difficult to optimize their fluorescence properties, which took approximately 15 years between the availability of the first useable CFP, enhanced cyan fluorescent protein (ECFP), and that of a variant with almost perfect fluorescence efficiency, mTurquoise2. To understand the molecular bases of the progressive improvement in between these two CFPs, we have studied by incoherent neutron scattering the dynamics of five different variants exhibiting progressively increased fluorescence efficiency along the evolution pathway. Our results correlate well with the analysis of the previously determined X-ray crystallographic structures, which show an increase in flexibility between ECFP and the second variant, Cerulean, which is then hindered in the three later variants, SCFP3A (Super Cyan Fluorescent Protein 3A), mTurquoise and mTurquoise2. This confirms that increasing the rigidity of the direct environment of the fluorescent chromophore is not the sole parameter leading to brighter fluorescent proteins and that increased flexibility in some cases may be helpful.</jats:p>

Analysis of theLeishmania mexicanapromastigote cell cycle using imaging flow cytometry provides new insights into cell cycle flexibility and events of short duration

“…However, to combine ACS fluorescence with mNG:KINF may, depending on its brightness, require a tag other than mCherry, which we found when tagged to KINF was not detectable by IFC. The recently developed mScarlet3 (63), or perhaps a cyan fluorescent protein (64) may provide an alternative that also permits co-staining and analysis with DCO.…”

Structure‐guided evolution of Green2 toward photostability and quantum yield enhancement by F145Y substitution

“…The seventh strand of the β‐barrel is critical for QY, as demonstrated by rational site‐directed mutagenesis on the backbone of cyan FPs. The QY of Super Cyan Fluorescent Protein 3A (QY = 0.56), which contains only the H148D mutation, is greater than that of Cerulean (QY = 0.49) that contains the two mutations Y145A and H148H 17 . This result indicates the critical role of Y145 in QY.…”

“…The QY of Super Cyan Fluorescent Protein 3A (QY = 0.56), which contains only the H148D mutation, is greater than that of Cerulean (QY = 0.49) that contains the two mutations Y145A and H148H. 17 This result indicates the critical role of Y145 in QY. To disclose the potential mechanism of Y145 in the photophysical and photochemical properties, here we present a systematic study investigating the contributions of electrostatics to energetic features of the chromophore's potential energy surface for eliminating the barrier between the ground and excited states.…”

Uncovering the Dynamics of Confined Water Using Neutron Scattering: Perspectives

“…However, the advent of such research has just begun due to the development of the third-generation neutron backscattering instruments [4,30,[46][47][48], but continued growth will depend on the highest-flux and flexibility provided by the backscattering spectrometer MIRACLES. Additionally, neutron scattering offers the unique capability of performing experiments in various extreme environments, such as humidity [49], pressure [50], electrical stimuli [51], pump probe [52].…”