We probe the transport properties in protein solutions stable with respect to any, solid or liquid, phase separation as a step in the understanding of transport in the cytosol of live cells. We determine the mean-squared displacement of probe particles in the time range 10;{-3}-10 s in solutions of a model protein. The tested solutions exhibit significant elasticity at high frequencies, while at low frequencies, they are purely viscous. We attribute this viscoelasticity to a dense network of weakly-bound chains of protein molecules with characteristic lifetime of 10-100 ms. The found intrinsic viscoelasticity of protein solutions should be considered in biochemical kinetics models.
The goal of our project is to determine and compare the binding thermodynamics of the interaction between β‐lactamase Inhibitor Protein (BLIP) mutants and Class A β‐Lactamases using ITC. We have previously determined the thermodynamics of binding interactions between TEM‐1, class A β‐lactamase family, and a set of alanine substituted contact residue mutants of BLIP (Wang J., et al. JBC. 2007; 282:17676–84). We have extended our approach to other members of class A β‐lactamases. Here, we report the binding thermodynamics between the inactive TEM‐1 mutant (S70A) and BLIP. We find that the alanine substitution of the active site Ser70 of TEM‐1 results in the lowering of the iso‐enthalpic temperature, which is more enthalpically favorable. The binding between BLIP and Bla1, another class A β‐lactamase, is dominated by an entropy driven force with an extrapolated iso‐enthalpic temperature of 42oC. Its binding heat capacity change is ∼ −450 cal/mol.K (∼ 20% less negative than that of BLIP/TEM‐1). However, the BLIP mutant W150A interaction with Bla1 shows a non‐linear relationship between the binding enthalpy and temperature with a positive binding heat capacity change at low temperature and a negative binding heat capacity change at high temperature, which is contrary to the general observation that the heat capacity change is negative for protein‐protein interactions.Support from AHA & UH to D.C. and NIH AI32956 to T.P.
The S. clavuligerus ß‐Lactamase Inhibitor Protein (BLIP) is an effective inhibitor of class A ß‐lactamases, including the TEM‐1 β‐Lactamase (TEM1). A previous report from our groups shows that Y143A mutant of BLIP binds to TEM1 with similar affinity as the wild type, but with a larger entropy contribution to binding. The Y50A mutant binds to TEM1 50 fold stronger than wild type, which is the result of increased enthalpy driving forces (Wang J., et al. JBC. 2007; 282:17676–84). In addition, binding of BLIP Y50A with TEM1 exhibits a less negative ΔCp than that of the wild type complex. These findings suggest that binding between BLIP Y50A and TEM1 is enhanced by hydrophilic interactions. Interestingly, our stopped‐flow kinetic measurements show that the association rate of BLIP Y50A with TEM1 is similar to that of the wild type, demonstrating the increased affinity is mainly due to the slower dissociation. To understand the changes in binding thermodynamics and kinetics, we initiated structural studies of these complexes. After extensive crystallization screens, some high quality crystals were obtained for the BLIP Y50A/TEM1 complex and the BLIP Y143A/TEM1 complex crystallized in micro‐needle form. We are currently optimizing conditions to obtain high‐quality crystals. Future X‐ray diffraction analysis will determine if the crystals are suitable for structure determination.Support from AHA & UH to D.C. and NIH to T.P.
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