Structural guidelines for stabilization of α-helical coiled coils via PEG stapling.

Macrocyclization or stapling is one of the most well-known and generally applicable strategies for enhancing peptide/protein conformational stability and target binding affinity. However, there are limited structure- or sequence-based guidelines for the incorporation of optimal interhelical staples within coiled coils: the location and length of an interhelical staple is either arbitrarily chosen or requires significant optimization. Here we explore the impact of interhelical PEG stapling on the conformational stability and proteolytic resistance of a model disulfide-bound heterodimeric coiled coil. We demonstrate that (1) interhelical PEG staples are more stabilizing when placed farther from an existing disulfide crosslink; (2) e/g' staples are more stabilizing than f/b' or b/c' staples; (3) PEG staples between different positions have different optimal staple lengths; (4) PEG stapling tolerates variation in the structure of the PEG linker and in the mode of conjugation; and (5) the guidelines developed here enable the rational design of a stabilized PEG-stapled HER-2 affibody with enhanced conformational stability and proteolytic resistance.

Prerequisites for Stabilizing Long-Range Synergistic Interactions among b-, c-, and f-Residues in Coiled Coils.

Coiled coils are among the most abundant tertiary and quaternary structures found in proteins. A growing body of evidence suggests that long-range synergistic interactions among solvent-exposed residues can contribute substantially to coiled-coil conformational stability, but our understanding of the key sequence and structural prerequisites of this effect is still developing. Here, we show that the strength of synergistic interaction involving a b-position Glu (i), an f-position Tyr (i + 4), and a c-position Lys (i + 8) depends on the identity of the f-position residue, the length and stability of the coiled coil, and its oligomerization stoichiometry/surface accessibility. Combined with previous observations, these results map out predictable sequence- and structure-based criteria for enhancing coiled-coil stability by up to -0.58 kcal/mol per monomer (or -2.32 kcal/mol per coiled-coil tetramer). Our observations expand the available tools for enhancing coiled coil stability by sequence variation at solvent-exposed b-, c-, and f-positions and suggest the need to exercise care in the choice of substitutions at these positions for application-specific purposes.

PEGylation Increases the Strength of a Nearby NH-π Hydrogen Bond in the WW Domain.

Here we show that an NH-π interaction between a highly conserved Asn and a nearby Trp stabilizes the WW domain of the human protein Pin1. The strength of this NH-π interaction depends on the structure of the arene, with NH-π interactions involving Trp or naphthylalanine being substantially more stabilizing than those involving Tyr or Phe. Calculations suggest arene size and polarizability are key structural determinants of NH-π interaction strength. Methylation or PEGylation of the Asn side-chain amide nitrogen each strengthens the associated NH-π interaction, though likely for different reasons. We hypothesize that methylation introduces steric clashes that destabilize conformations in which the NH-π interaction is not possible, whereas PEGylation strengthens the NH-π interaction via localized desolvation of the protein surface.

Inhibition of 14-3-3 Proteins Alters Neural Oscillations in Mice.

Accumulating evidence suggests that schizophrenia is a disorder of the brain's communication, a result of functional and structural dysconnectivities. Patients with schizophrenia exhibit irregular neuronal circuit and network activity, but the causes and consequences of such activity remain largely unknown. Inhibition of 14-3-3 proteins in the mouse brain leads to the expression of multiple schizophrenia endophenotypes. Here we investigated how 14-3-3 inhibition alters neuronal network activity in the mouse hippocampus (HPC) and prefrontal cortex (PFC), key brain regions implicated in schizophrenia pathophysiology. We implanted monopolar recording electrodes in these two regions to record local field potentials both at rest and during a cognitive task. Through our assessment of band power, coherence, and phase-amplitude coupling, we found that neural oscillations in the theta and gamma frequency ranges were altered as a result of 14-3-3 dysfunction. Utilizing transgenic and viral mouse models to assess the effects of chronic and acute 14-3-3 inhibition on oscillatory activities, respectively, we observed several fundamental similarities and differences between the two models. We localized viral mediated 14-3-3 protein inhibition to either the HPC or PFC, allowing us to assess the individual contributions of each region to the observed changes in neural oscillations. These findings identify a novel role of 14-3-3 proteins in neural oscillations that may have implications for our understanding of schizophrenia neurobiology.

Long-range PEG Stapling: Macrocyclization for Increased Protein Conformational Stability and Resistance to Proteolysis.

We previously showed that long-range stapling of two Asn-linked O-allyl PEG oligomers via olefin metathesis substantially increases the conformational stability of the WW domain through an entropic effect. The impact of stapling was more favorable when the staple connected positions that were far apart in primary sequence but close in the folded tertiary structure. Here we validate these criteria for identifying new stabilizing PEG-stapling sites within the WW domain and the SH3 domain, both ß-sheet proteins. We find that stapling via olefin metathesis vs. the copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) results in similar energetic benefits, suggesting that olefin and triazole staples can be used interchangeably. Proteolysis assays of selected WW variants reveal that the observed staple-based increases in conformational stability lead to enhanced proteolytic resistance. Finally, we find that an intermolecular staple dramatically increases the quaternary structural stability of an α-helical GCN4 coiled-coil heterodimer.

Multiple organ dysfunction and systemic inflammation after spinal cord injury: a complex relationship.

Spinal cord injury (SCI) is a devastating event that results in significant physical disabilities for affected individuals. Apart from local injury within the spinal cord, SCI patients develop a variety of complications characterized by multiple organ dysfunction or failure. These disorders, such as neurogenic pain, depression, lung injury, cardiovascular disease, liver damage, kidney dysfunction, urinary tract infection, and increased susceptibility to pathogen infection, are common in injured patients, hinder functional recovery, and can even be life threatening. Multiple lines of evidence point to pathological connections emanating from the injured spinal cord, post-injury systemic inflammation, and immune suppression as important multifactorial mechanisms underlying post-SCI complications. SCI triggers systemic inflammatory responses marked by increased circulation of immune cells and pro-inflammatory mediators, which result in the infiltration of inflammatory cells into secondary organs and persistence of an inflammatory microenvironment that contributes to organ dysfunction. SCI also induces immune deficiency through immune organ dysfunction, resulting in impaired responsiveness to pathogen infection. In this review, we summarize current evidence demonstrating the relevance of inflammatory conditions and immune suppression in several complications frequently seen following SCI. In addition, we highlight the potential pathways by which inflammatory and immune cues contribute to multiple organ failure and dysfunction and discuss current anti-inflammatory approaches used to alleviate post-SCI complications. A comprehensive review of this literature may provide new insights into therapeutic strategies against complications after SCI by targeting systemic inflammation.

Sphingolipids in spinal cord injury.

Spinal cord injury (SCI) is a debilitating condition that affects millions of individuals worldwide. Despite progress over the last few decades, the molecular mechanisms of secondary SCI that continue to occur days and weeks after the original trauma remain poorly understood. As a result, current therapies for SCI are only marginally effective. Sphingolipids, a diverse class of bioactive lipids, have been shown to regulate SCI repair and key secondary injury processes such as apoptosis, ischemia and inflammation. This review will discuss the numerous roles of sphingolipids and highlight the potential of sphingolipid-targeted therapies for SCI.