α-proton Chemical Shift Index and Amide Proton Chemical Shift Temperature Coefficient of Melittin in Methanol: Indicators for a Helix Structure and an Intra-Molecular Hydrogen Bond?

The methods of the α-proton chemical shift index (CSI) and the amide proton (NH) chemical shift temperature coefficient (Δδ/ΔT) were found experimentally by a number of studies on proton NMR chemical shifts of peptides and proteins in an aqueous solution, and have been widely accepted. They provide an insight into secondary structures and intra-molecular hydrogen bonds in peptides and proteins without complex calculation. Our question is whether the methods are applicable to helical peptides in methanol. Melittin, a peptide of 26 amino acid residues found in bee venom, has been known to consist of two helices (the residues 2-11 and 13-26) connected by a kink section (the residues 11-13). Employing the methods for melittin in methanol, we have shown that most of the CSI's for the residues 3-10 and 14-26 are - 1, and the Δδ/ΔT's for the residues 5-26 except 6 and 14 are more positive than - 6 ppb/℃. If the methods are applicable to melittin in methanol, these results indicate that helix structures are formed in the regions of the residues 3-10 and 14-26 and NH's in the residues 5-26 except 6 and 14 are involved in intra-molecular hydrogen bonds. The helical structure evaluated from the methods, hence, agrees nearly with the known helix structure. We also apply the methods to D-Pro14 melittin (synthetic melittin) and alamethicin (a peptide of 20 amino acid residues) in methanol, and show that they virtually work well.

1H, 13C and 15N backbone NMR chemical shift assignments of the C-terminal P4 domain of Ahnak.

Ahnak is a ~ 700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca2+ signaling in cardiomyocytes by interacting with the ß subunit of the L-type Ca2+ channel (CaV1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete 1H, 13C and 15N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the CaV1.2-Ahnak complex.

Backbone chemical shift assignments and secondary structure analysis of the U1 protein from the Bas-Congo virus.

The Bas-Congo virus (BASV) is the first rhabdovirus associated with a human outbreak of acute hemorrhagic fever. The single-stranded, negative-sense RNA genome of BASV contains the five core genes present in all rhabdoviral genomes plus an additional three genes, annotated U1, U2, and U3, with weak (<21%) sequence similarity only to a handful of genes observed in a few other rhabdoviral genomes. The function of the rhabdoviral U proteins is unknown, but, they are hypothesized to play a role in viral infection or replication. To better understand this unique family of proteins, a construct containing residues 27-203 of the 216-residue U1 protein (BASV-U1*) was prepared. By collecting data in 0.5 M urea it was possible to eliminate transient association enough to enable the assignment of most of the observable 1HN, 1Hα, 15N, 13Cα, 13Cß, and 13C´ chemical shifts for BASV-U1* that will provide a foundation to study its solution properties. The analyses of these chemical shifts along with 15N-edited NOESY data enabled the identification of the elements of secondary structure present in BASV-U1*.

Type 2 ryanodine receptor domain A contains a unique and dynamic α-helix that transitions to a ß-strand in a mutant linked with a heritable cardiomyopathy.

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca(2+)) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca(2+) sensitivity, propagating sarcoplasmic reticulum luminal Ca(2+) release in the process of excitation-contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1-559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the ß-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense "columns" projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.