Preview : Structure of G-CaMP2

A high-resolution crystal structure of the genetically-encoded calcium indicator G-CaMP2 would aid in rational design of improved calcium indicators. Crystallization of G-CaMP2 was first reported here :

Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

M. M. Rodríguez Guilbe, E. C. Alfaro Malavé, J. Akerboom, J. S. Marvin, L. L. Looger and E. R. Schreiter

Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way.

High-resolution atomic structures and mutational analysis were presented at SfN 2008 (see this previous post)

However, today a competing group has published an independent report on a similar set of G-CaMP2 structures in Cell Structure.  More details to come…

Structural Basis for Calcium Sensing by GCaMP2

Qi Wang¹,Bo Shui²,Michael I. Kotlikoff²andHolger Sondermann^1,,

Genetically encoded Ca²⁺ indicators are important tools that enable the measurement of Ca²⁺ dynamics in a physiologically relevant context. GCaMP2, one ofthe most robust indicators, is a circularly permutated EGFP (cpEGFP)/M13/calmodulin (CaM) fusion protein that has been successfully used for studying Ca²⁺ fluxes invivo in the heart and vasculature of transgenic mice. Here we describe crystal structures of bright and dim states of GCaMP2 that reveala sophisticated molecular mechanism for Ca²⁺ sensing. In the bright state, CaM stabilizes the fluorophore in an ionized state similar to that observed in EGFP. Mutational analysis confirmed critical interactions between the fluorophore and elements of the fused peptides. Solution scattering studies indicate thatthe Ca²⁺-free form of GCaMP2 is a compact, predocked state, suggesting a molecular basis for the relatively rapid signaling kinetics reported for this indicator. These studies provide a structural basis for the rational design of improved Ca²⁺-sensitive probes.

Some interesting posters @ SfN

Here’s a few posters that caught my eye at SfN.  Click the meeting planner for the full abstract

Optimizing two-photon activation of channelrhodopsin-2 for stimulation at cellular resolution

J. P. RICKGAUER^1,2, D. W. TANK^1,2; 

Spiral pattern of 2-photon excitation can drive neurons to spike.  A low NA objective helps. Need to do piezo-based Z-scanning if you use high NA, don’t with low NA.

In vivo two-photon imaging 1 mm deep into cortical brain tissue with novel microprism probe 

*T. H. CHIA, M. J. LEVENE; 

A cute method to image 1mm into cortex with 2-photon imaging. They used 2-6 month old mice. The just took a triangular prism whose hypotenuse was silvered and stuck it in the cortex. Then they internally reflected the beam off the prism and fired it sideways into cortex. Got good SNR to 300um lateral distance.  Some clippling of beam at edges of the prism gave somewhat inconsistent spatial resolution.

Self-complementary adeno-associated viral vectors for fast, efficient labeling of neurons and astrocytes in visual cortex in vivo

R. L. LOWERY¹, Y. ZHANG², C. LAMANTIA¹, B. K. HARVEY³, A. K. MAJEWSKA¹; 

AAV is the way to go for expression of GECIs and ChR2 in vivo, but it takes a long time to express at high levels (2 weeks). They show that using a double stranded DNA version of AAV rather than single stranded gets protein expression up high much faster. Very high expression after one week. This is because the virus doesn’t need to take the time to make the second strand before expressing the protein.  See Xiao, X J. Virol 1998

Detection of single action potentials in vitro and in vivo with genetically-encoded Ca²⁺ sensors

S. MEYER ZUM ALTEN BORGLOH¹, D. J. WALLACE², S. ASTORI³, Y. YANG³, M. BAUSEN³, S. KUGLER⁴, M. MANK⁵, O. GRIESBECK⁵, J. NAKAI⁶, A. MIYAWAKI⁶, A. E. PALMER⁷, R. Y. TSIEN⁷, R. SPRENGEL³, J. N. D. KERR², W. DENK³, M. T. HASAN³; 

Everything in the poster was in the Nature Methods paper.  Conversation reveled that YC3.60 works as well or better than D3cpv. Only have done up to whisker evoked stimulation, no imaging of spontaneous YC3.60 signals yet.

Characterization of improved probes for the hybrid voltage sensor method of voltage imaging

D. WANG¹, Z. ZHANG², B. CHANDA¹, M. B. JACKSON¹; 

A nice little sensor optimization poster.  They took the hVOS hybrid voltage sensor of dipicrylamine with membrane tethered GFP and improved it by changing the chromophore to Cerulean, and by using the “membrane-staple” strategy. Having membrane anchors on both the N and C-termini gave better quenching. Fast response, ~0.5ms, and 20% dF/F.

Crystal structure of the genetically encoded calcium indicator gcamp2

*J. AKERBOOM¹, L. TIAN¹, S. VISWANATHAN¹, S. A. HIRES¹, J. S. MARVIN¹, E. R. SCHREITER², L. L. LOOGER¹; 

Jasper made crystal structures of G-CaMP2 in the apo and bound states.  Bound states crystalized as a heterodimer, but he was able to also crystalize the monomer. The structures show a pore to the chromophore in the apo state that is plugged in the Ca-bound state. Thus, the quenched apo state is due to solvent access to the chromophore.  This structural data should help rational design of better G-CaMP sensors.