The synthetic antimicrobial peptide CM15, a hybrid of N-terminal sequences from cecropin and melittin peptides, has been shown to be extremely potent. Its mechanism of action has been speculated to involve pore formation based on prior site-directed spin labeling studies. This study examines four single-site β-thiocyanatoalanine variants of CM15 in which the artificial amino acid side chain acts as a vibrational reporter of its local environment through the frequency and lineshape of the unique CN stretching band in the infrared spectrum. Circular dichroism experiments indicate that the placements of the artificial side chain have only small perturbative effects on the membrane-bound secondary structure of the CM15 peptide. All variant peptides were placed in buffer solution, in contact with dodecylphosphatidylcholine micelles, and in contact with vesicles formed from E. coli polar lipid extract. At each site, the CN stretching band reports a different behavior. Time-dependent attenuated total reflectance infrared spectra were also collected for each variant as it was allowed to remodel the E. coli lipid vesicles. These experiments agree with the previously proposed formation of toroidal pores, in which each peptide finds itself in an increasingly homogeneous and curved local environment without apparent peptide-peptide interactions. This work also demonstrates the excellent sensitivity of the SCN stretching vibration to small changes in peptide-lipid interfacial structure. Keywordsantimicrobial peptides; cyanylated cysteine; nitrile vibrations; membrane-active peptides; sitedirected labeling; toroidal pores Characterizing the structure, structural distribution, and membrane binding geometry of peripheral membrane proteins (PMPs) is a challenge that requires new experimental approaches. Recent advances in crystallography using surfactants(1) and solid state NMR of oriented samples(2) have led to new insights into the structure and function of a number of such proteins and peptides. However, a hallmark of these studies is that the experimental approach used depends to a large extent on the lipid and protein system of interest without a single, unified approach that is applicable to proteins of any size in arbitrary lipid systems. EPR spectroscopy of site-directed spin labels has this sought-after flexibility when applied to PMPs,(3) with documented limitations due to the size and chemical nature of the most typical spin label(4) and the need for external solution-or lipid-phase paramagnetic species to address directly the extent of membrane burial.(3) Antimicrobial peptides (AMPs) are a subset of membrane-active species generating much recent interest due to their possible use as antibiotic agents in an era of global overuse of antibiotic drugs.(5) The relative simplicity * to whom correspondence may be addressed; telephone 610-896-1217, fax 610-896-4963, clonderg@haverford.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 December 27. NIH-PA Author Manusc...

To investigate the cyanylated cysteine vibrational probe group’s ability to report on binding-induced changes along a protein–protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques.

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The aggregation of amyloidogenic proteins is infamous for being highly chaotic, with small variations in conditions leading to large changes in aggregation rates. We present experimental evidence that the origin of this phenomenom for a broad variety of amyloidogneic oligomers is related to the critical concentration for the formation of particular type of prefibrillar oligomer. Using chemical probes and real time solution NMR, we show that oligomers of this type reversibly form at sharply defined critical concentrations and temperatures in a manner similar to a phase transition. Similarly, the kinetics of fiber formation also show strong non-linearity near these critical points. For some amyloidogenic proteins such as IAPP, the pathway of amyloid formation switches as the critical point is approached, with aggregation initiating at different regions of the protein above and below the critical point. We also show that part of the effect of some inhibitors on amyloid aggregation is to abolish or shift the critical concentration, and map the interface between monomeric IAPP and Ab and these aggregates. 261-Pos Board B30 Atomic Resolution Insights into the Aggregation of the Murine Prion Protein by NMR

Intrinsically disordered proteins (IDPs) that undergo a coupled folding and binding event are found to be important in many recognition and signaling processes. However, exactly how this mechanism is linked to these functions is not completely understood. This is primarily because the structural flexibility of IDPs limits the number of suitable characterization techniques. We are using small-angle neutron scattering (SANS) to investigate the structure and binding interaction properties between NCBD, an IDP region of CREB binding protein (CBP), and its binding partner, ACTR, which also is an IDP. CBP is a transcription co-activator that is essential in embryonic development, growth control, and homeostasis, and its dysfunction is implicated in neurological disorders such as Huntington's disease and some cancers. SANS indicates the NCBD/ ACTR complex is a globular, folded structure with a smaller radius of gyration compared to ACTR alone, which is mostly unfolded. Using ab initio shape reconstruction programs to gain further insight into structural flexibility, we find good agreement between the shape reconstruction and NMR structure for the NCBD/ACTR complex. In contrast, SANS reveals the nature of how ACTR is more expanded when in its unbound, unfolded state. This research should provide new possibilities for the study of disordered protein regions and yield unique perspectives into the mechanism of IDP function.