1) Rajanikanth V, Sharma AK, Rajyalakshmi M, Chandra K, Chary KVR, Sharma
Y.
Liaison between Myristoylation and Cryptic EF-Hand Motif Confers Ca(2+)
Sensitivity to Neuronal Calcium Sensor-1. Biochemistry. 2015 Jan 21. [Epub
ahead of print] PubMed PMID: 25565019.
Abstract:
Many members of the neuronal calcium sensor (NCS) protein family have a striking
coexistence of two characteristics, that is, N-myristoylation and the cryptic EF-1 motif. We
investigated the rationale behind this correlation in neuronal calcium sensor-1 (NCS-1) by
restoring Ca2+ binding ability of the disabled EF-1 loop by appropriate mutations. The
concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall
Ca2+affinity, conformational flexibility, and functional activation of downstream effecter
molecules (i.e., PI4Kβ). Of a particular note, Ca2+ induced conformational change (which is
the first premise for a CaBP to be considered as sensor) is considerably reduced in
myristoylated proteins in which Ca2+-binding to EF-1 is restored. Moreover, Ca2+, which
otherwise augments the enzymatic activity of PI4Kβ (modulated by NCS-1), leads to a
further decline in the modulated PI4Kβ activity by myristoylated mutants (with canonical EF1) pointing toward a loss of Ca2+ signaling and specificity at the structural as well as
functional levels. This study establishes the presence of the strong liaison between
myristoylation and cryptic EF-1 in NCS-1. Breaking this liaison results in the failure of
Ca2+ specific signal transduction to downstream effecter molecules despite Ca2+ binding.
Thus, the EF-1 disability is a prerequisite in order to append myristoylation signaling while
preserving structural robustness and Ca2+sensitivity/specificity in NCS-1.
2) Narayan S, Kombrabail MH, Das S, Singh H, Chary KVR, Rao BJ,
Krishnamoorthy G.
Site-specific fluorescence dynamics in an RNA 'thermometer' reveals the role of ribosome
binding in its temperature-sensitive switch function. Nucleic Acids Res. 2015 Jan
9;43(1):493-503. doi: 10.1093/nar/gku1264. Epub 2014 Dec 3. PubMed PMID: 25477380.
Abstract:
RNA thermometers control the translation of several heat shock and virulence genes by
their temperature-sensitive structural transitions. Changes in the structure and dynamics of
MiniROSE RNA, which regulates translation in the temperature range of 20-45°C, were
studied by site specifically replacing seven adenine residues with the fluorescent analog, 2aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs
were compared in their free versus ribosome-bound states for the first time. Noticeably,
position dependence of fluorescence observables, which was prominent at 20°C, was
persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC.
Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant
at 45°C was ablated in ribosome-bound state, when compared to those at 20°C,
underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant
increase in the value of mean lifetime for 2-AP corresponding to Shine-Dalgarno sequences,
when the temperature was raised from 20 to 45°C, to values seen in the presence of urea
at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only
when it was ribosome bound. Taken all together, we propose a model where we invoke that
ribosome binding of the RNA thermometer critically regulates temperature sensing functions
in MiniROSE RNA.
II. Dr. Sunita Patel received the Best Poster award
Patel S, Chary KVR, "Structure and dynamics of an intrinsically order and disordered
proteins" in the symposium on "Brainstorming Meeting on Proteomics: Present and Future
held at Centre for Cellular and Molecular Biology, Hyderabad from 22-24 November 2014.