Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates.

The ClpS adaptor collaborates with the AAA+ ClpAP protease to recognize and degrade N-degron substrates. ClpS binds the substrate N-degron and assembles into a high-affinity ClpS-substrate-ClpA complex, but how the N-degron is transferred from ClpS to the axial pore of the AAA+ ClpA unfoldase to initiate degradation is not known. Here we demonstrate that the unstructured N-terminal extension (NTE) of ClpS enters the ClpA processing pore in the active ternary complex.

Surface electrostatics of lipid bilayers by EPR of a pH-sensitive spin-labeled lipid.

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids' polar headgroup.

Prevention of benzyl alcohol-induced aggregation of chymotrypsinogen by PEGylation.

Objectives: Addition of the antimicrobial preservative benzyl alcohol to reconstitution buffer promotes the formation of undesirable aggregates in multidose protein formulations. Herein we investigated the efficiency of PEGylation (attachment of poly(ethylene glycol)) to prevent benzyl alcohol-induced aggregation of the model protein α-chymotrypsinogen A (aCTgn).

Methods: Various PEG-aCTgn conjugates were prepared using PEG with a molecular weight of either 700 or 5000 Da by varying the PEG-to-protein ratio during synthesis and the formation of insoluble aggregates was studied.

The IbpA and IbpB small heat-shock proteins are substrates of the AAA+ Lon protease.

Small heat-shock proteins (sHSPs) are a widely conserved family of molecular chaperones, all containing a conserved alpha-crystallin domain flanked by variable N- and C-terminal tails. We report that IbpA and IbpB, the sHSPs of Escherichia coli, are substrates for the AAA+ Lon protease. This ATP-fueled enzyme degraded purified IbpA substantially more slowly than purified IbpB, and we demonstrate that this disparity is a consequence of differences in maximal Lon degradation rates and not in substrate affinity.

Enzymatic activity and thermal stability of PEG-alpha-chymotrypsin conjugates.

Alpha-chymotrypsin was chemically modified with methoxypoly(ethylene glycol) (PEG) of different molecular weights (700, 2,000, and 5,000 Da) and the amount of polymer attached to the enzyme was varied systematically from 1 to 9 PEG molecules per enzyme molecule. Upon PEG conjugation, enzyme catalytic turnover (k (cat)) decreased by 50% and substrate affinity was lowered as evidenced by an increase in the K (M) from 0.05 to 0.

Stabilization of alpha-chymotrypsin upon PEGylation correlates with reduced structural dynamics.

Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. Poly(ethylene glycol) (PEG) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. The observed increase in thermodynamic stability of several proteins upon PEGylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing.