Anna Á. Rauscher

Relating the shape of protein binding sites to binding affinity profiles: is there an association?

BackgroundVarious pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values) and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs) were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses.ResultsPCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors.ConclusionsThis is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that, except for few specific cases, the shapes of the binding pockets have relatively low weights in the determination of the affinity profiles of proteins. Since the MAF profile is closely related to the target specificity of ligand binding sites we can conclude that the shape of the binding site is not a pivotal factor in selecting drug targets. Nonetheless, based on strong specific associations between certain MAF profiles and specific geometric descriptors we identified, the shapes of the binding sites do have a crucial role in virtual drug design for certain drug categories, including morphine derivatives, benzodiazepines, barbiturates and antihistamines.

Internal friction in enzyme reactions.

The empirical concept of internal friction was introduced 20 years ago. This review summarizes the results of experimental and theoretical studies that help to uncover the nature of internal friction. After the history of the concept, we describe the experimental challenges in measuring and interpreting internal friction based on the viscosity dependence of enzyme reactions. We also present speculations about the structural background of this viscosity dependence. Finally, some models about the relationship between the energy landscape and internal friction are outlined. Alternative concepts regarding the viscosity dependence of enzyme reactions are also discussed. ©2012 IUBMB Life, 65(1):35–42, 2013

Magnesium modulates actin binding and ADP release in myosin motors

Background: Magnesium may be an important physiological regulator of myosin motor activity. Results: Mg2+ inhibits the ADP release rate constant in the subset of myosins examined and reduces actin affinity in the post-hydrolysis state in myosin V. Conclusion: Mg2+ alters contractile velocity without altering overall tension-generating capacity. Significance: Mg2+-dependent regulation of motor activity is conserved in myosin motors. We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, β-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg2+-dependent manner (0.3–9.0 mm free Mg2+) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg2+ in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg2+ in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg2+ coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg2+ concentrations, demonstrating that the ADP release rate constant is slowed by Mg2+ in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg2+ reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg2+ inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg2+-dependent alterations in actin binding. Overall, our results suggest that Mg2+ reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.

Temperature dependence of internal friction in enzyme reactions

Our aim was to elucidate the physical background of internal friction of enzyme reactions by investigating the temperature dependence of internal viscosity. By rapid transient kinetic methods, we directly measured the rate constant of trypsin 4 activation, which is an interdomain conformational rearrangement, as a function of temperature and solvent viscosity. We found that the apparent internal viscosity shows an Arrhenius‐like temperature dependence, which can be characterized by the activation energy of internal friction. Glycine and alanine mutations were introduced at a single position of the hinge of the interdomain region to evaluate how the flexibility of the hinge affects internal friction. We found that the apparent activation energies of the conformational change and the internal friction are interconvertible parameters depending on the protein flexibility. The more flexible a protein was, the greater proportion of the total activation energy of the reaction was observed as the apparent activation energy of internal friction. Based on the coupling of the internal and external movements of the protein during its conformational change, we constructed a model that quantitatively relates activation energy, internal friction, and protein flexibility.—Rauscher, A. Á., Simon, Z., Szöllősi, G. J., Gráf, L., Derényi, I., Malnasi‐Csizmadia, A. Temperature dependence of internal friction in enzyme reactions. FASEB J. 25, 2804‐2813 (2011). www.fasebj.org

Targeting Myosin by Blebbistatin Derivatives: Optimization and Pharmacological Potential

Blebbistatin is a widely used inhibitor of myosin 2 that enables the study of a broad range of cytoskeleton-related processes. However, blebbistatin has several limitations hindering its applicability: it is fluorescent, poorly water soluble, cytotoxic, and prone to (photo)degradation. Despite these adverse effects, being the only available myosin 2-specific inhibitor, blebbistatin is rather a choice of necessity. Blebbistatin has been modified to improve its properties and some of the new compounds have proven to be useful replacements of the original molecule. This review summarizes recent results on blebbistatin development. We also discuss the pharmacological perspectives of these efforts, as myosins are becoming promising drug target candidates for a variety of conditions ranging from neurodegeneration to muscle disease, wound healing, and cancer metastasis.

Viscosity dependence of passage through a fluctuating bottleneck

We generalize the model of a rate process involving the passage of an object through a fluctuating bottleneck. The rate of passage is considered to be proportional to a power function of the radius of the bottleneck with exponent α > 0. The fluctuations of the bottleneck are coupled to the motion of the surrounding medium and governed by Langevin dynamics. We show numerically and also explain analytically that for slow bottleneck fluctuations the long time decay rate of the process has a fractional power law dependence on the solvent viscosity with exponent α/(α + 2). The results are consistent with the experimental data on ligand binding to myoglobin, and might also be relevant to other reactions for which exponents between 0 and 1 were reported.

Relationship Between Internal Friction and the Roughness of the Energy Landscape of Protein Conformational Changes

We constructed a quantitative model based on experimental data that describes the relationship between the roughness of the energy landscape, activation energy and internal friction of enzyme conformational changes. We investigated an interdomain conformational rearrangement, trypsinogen 4 activation using transient kinetic methods. The temperature and viscosity dependence of the rate constant of the conformational change was measured in order to determine the temperature dependence of its internal friction. To test the effect of flexibility on internal friction, glycine and alanine mutations at a single position of the hinge of the interdomain region were introduced. Internal friction showed an Arrhenius-like temperature dependence, the characteristic energy of which increased with the flexibility of the hinge.We found that the activation energy, i.e. the height of the energy landscape, is partially converted into internal friction to an extent depending on the flexibility of the protein. We interpret this phenomenon using a model that assumes different hierarchical levels of roughness of the energy landscape.

Protein Flexibility Partitions the Effects of Energy Landscape Roughness Between Activation Energy and Internal Friction

The rate of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. By investigating the internal friction during the activation of two trypsin mutants at various temperatures and external viscosities we have discovered that the temperature dependence of the internal friction shows an Arrhenius-like behavior. The characteristic energy of the Arrhenius formula, however, can change dramatically upon the replacement of a single amino acid at a hinge position (thereby affecting the flexibility of the protein), or by crossing a critical temperature. At the same time, the activation energy of the conformational transition also changes with a similar magnitude, but in the opposite direction. These observations shed light on the intricate interplay between the apparent internal friction and activation energy. Moreover, we have found that the more flexible a protein is the greater proportion of its activation energy is partitioned into internal friction. All these results have allowed us to come to the general conclusion that the different hierarchical levels of the roughness of the energy landscape along a conformational transition can be observed as either activation energy or internal friction depending on the degree of flexibility of the protein.