Structural Investigation of alphaB-Crystallin Core Domains
EMSL Project ID
16115
Abstract
Small Heat Shock Proteins (sHSPs) are ubiquitous cellular proteins that are over-expressed under conditions of stress in various organisms from bacteria to humans. ?B-Crystallin (?B), considered the archetypal sHSP, was originally found as an abundant eye-lens protein. ?B is also found in high levels in cardiac and skeletal muscles and in brain. Despite a growing list of biological roles for ?B, the structure and mechanism of its cyto-protective function is poorly understood. ?B forms supramolecular assemblies containing a heterogeneous population of oligomers. Hence, determination of the crystal structure of ?B has proved elusive for several decades. Employing a reductionist approach, a conserved, 89-residue core domain (common to all sHSPs and called the ?-crystallin domain) from human ?B is being investigated in our lab by solution-state NMR. We call this core domain ?B10.1 (the suffix refers to monomeric molecular weight). We have that a deamidated variant, N146D ?B10.1, has superior spectral properties highly suitable for solution state NMR. The variant and the wt core domain are similar: they both form dimers and they have nearly identical 1H-15N-HSQC spectra. Hence, we will first determine the high-resolution structure of N146D ?B10.1 and refine the structure of wt ?B10.1 from the variant structure. We will be able to collect sufficient data to calculate the structure of the protomer in the current PNNL NMR time awarded to us. We request one week of 600 MHZ of time (with the cryoprobe) to run experiments for observing inter-subunit NOEs. A human inherited missense mutation, R120G, is found in the core domain of ?B. Carriers of the mutant gene develop adult-onset desmin-related myopathy, with symptoms including cardiomyopathy and cataracts. We plan to determine the structure of the R120G mutant core domain by solution state NMR in order to understand ?B dysfunction. Towards this end, we request (i) one week of 800 MHz, (ii) one week of 600 MHz with cryoprobe, (iii) one week of 600 MHz (can be without cryoprobe), and (iv) one week of 900 MHz spectrometer times.
Project Details
Project type
Capability Research
Start Date
2005-10-01
End Date
2006-11-21
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members