Andrew D. Robertson

Very fast empirical prediction and rationalization of protein pKa values

A very fast empirical method is presented for structure‐based protein pKa prediction and rationalization. The desolvation effects and intra‐protein interactions, which cause variations in pKa values of protein ionizable groups, are empirically related to the positions and chemical nature of the groups proximate to the pKa sites. A computer program is written to automatically predict pKa values based on these empirical relationships within a couple of seconds. Unusual pKa values at buried active sites, which are among the most interesting protein pKa values, are predicted very well with the empirical method. A test on 233 carboxyl, 12 cysteine, 45 histidine, and 24 lysine pKa values in various proteins shows a root‐mean‐square deviation (RMSD) of 0.89 from experimental values. Removal of the 29 pKa values that are upper or lower limits results in an RMSD = 0.79 for the remaining 285 pKa values. Proteins 2005.

Empirical relationships between protein structure and carboxyl pKa values in proteins

Relationships between protein structure and ionization of carboxyl groups were investigated in 24 proteins of known structure and for which 115 aspartate and 97 glutamate pKa values are known. Mean pKa values for aspartates and glutamates are ≤ 3.4 (±1.0) and 4.1 (±0.8), respectively. For aspartates, mean pKa values are 3.9 (±1.0) and 3.1 (±0.9) in acidic (pI < 5) and basic (pI > 8) proteins, respectively, while mean pKa values for glutamates are approximately 4.2 for acidic and basic proteins. Burial of carboxyl groups leads to dispersion in pKa values: pKa values for solvent‐exposed groups show narrow distributions while values for buried groups range from < 2 to 6.7. Calculated electrostatic potentials at the carboxyl groups show modest correlations with experimental pKa values and these correlations are not improved by including simple surface‐area‐based terms to account for the effects of desolvation. Mean aspartate pKa values decrease with increasing numbers of hydrogen bonds but this is not observed at glutamates. Only 10 pKa values are > 5.5 and most are found in active sites or ligand‐binding sites. These carboxyl groups are buried and usually accept no more than one hydrogen bond. Aspartates and glutamates at the N‐termini of helices have mean pKa values of 2.8 (±0.5) and 3.4 (±0.6), respectively, about 0.6 units less than the overall mean values. Proteins 2002;48:388–403.

Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting “receptors,” the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.

Some thermodynamic implications for the thermostability of proteins

An analysis of the thermodynamics of protein stability reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. To a reasonable approximation, the temperature of maximal stability for the set of globular, water‐soluble proteins surveyed by Robertson and Murphy occurs at T* ∼283K, independent of the heat denaturation temperature, Tm. This observation indicates, at least for these proteins, that thermostability tends to be achieved through elevation of the stability curve rather than by broadening or through a horizontal shift to higher temperatures. The relationship between the free energy of maximal stability and the temperature of heat denaturation is such that an increase in maximal stability of ∼0.008 kJ/mole/residue is, on average, associated with a 1°C increase in Tm. An estimate of the energetic consequences of thermal expansion suggests that these effects may contribute significantly to the destabilization of the native state of proteins with increasing temperature.

The Determinants of Carboxyl pKa Values in Turkey Ovomucoid Third Domain

A computational methodology for protein pKa predictions, based on ab initio quantum mechanical treatment of part of the protein and linear Poisson–Boltzmann equation treatment of the bulk solvent, is presented. The method is used to predict and interpret the pKa values of the five carboxyl residues (Asp7, Glu10, Glu19, Asp27, and Glu43) in the serine protease inhibitor turkey ovomucoid third domain. All the predicted pKa values are within 0.5 pH units of experiment, with a root‐mean‐square deviation of 0.31 pH units. We show that the decreased pKa values observed for some of the residues are primarily due to hydrogen bonds to the carboxyl oxygens. Hydrogen bonds involving amide protons are shown to be particularly important, and the effect of hydrogen bonding is shown to be nonadditive. Hydrophobic effects are also shown to be important in raising the pKa. Interactions with charged residues are shown to have relatively little effect on the carboxyl pKa values in this protein, in general agreement with experiment. Proteins 2004;55:000–000.

Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.

Three-state Unfolding and Self-association of Maspin, a Tumor-suppressing Serpin

Maspin is a tumor suppressor protein expressed by normal human mammary epithelium but not by many breast tumor cell lines. Recombinant human maspin (rMaspin) inhibits tumor cell motility, invasion, and metastasis and thus has potential value as an anti-cancer therapeutic. Maspin is a member of the serpin family and, although the molecular mechanism by which maspin acts is unknown, recent work suggests that tissue plasminogen activator is a potential target. A puzzling observation in previous cell culture studies was loss of rMaspin activity at higher protein concentrations. One hypothesis to explain these results is self-association of rMaspin at the higher concentrations, which would be consistent with the tendency of serpins to form noncovalent polymers. This hypothesis is addressed by examining the relationship between rMaspin stability and self-association. Urea denaturation of rMaspin at pH 7 and 25 °C and at protein concentrations ranging from 0.01 to 0.2 mg/ml has been monitored by circular dichroism and intrinsic tryptophan fluorescence. Denaturation profiles show a protein concentration dependence and indicate the presence of at least one unfolding intermediate. The results suggest that destabilization of native monomeric rMaspin leads to partial unfolding and formation of an intermediate which can self-associate.

Kinetics and thermodynamics of conformational equilibria in native proteins by hydrogen exchange

Publisher Summary This chapter reviews that hydrogen exchange is a powerful and increasingly popular tool for the study of protein structure and dynamics. Proteins contain a number of functional groups bearing hydrogens that are labile to exchange with solvent hydrogens. The chemistry of these exchange or proton transfer events is well understood for small model compounds in which the functional groups are well exposed to solvent. However, when the exchange-labile hydrogen participates in a hydrogen bond or is otherwise excluded from solvent, then exchange can be slowed significantly. It discusses that the growing use of NH exchange is attributable to advances in both technology and in the understanding of the mechanisms by which NH exchange is modulated in proteins. The major technical advances are multidimensional nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). MS generally is used to measure the average exchange behavior of the protein as a whole or peptide fragments thereof. Many contributions in the understanding of the molecular mechanism of NH exchange have come from detailed analysis of NMR data. The chapter focuses on the execution and analysis of NMR experiments.

Structural biology: The ABC of a versatile engine

The ATP-binding cassette (ABC) transporter family of proteins all contain two nucleotide-binding domains (NBDs) along with two membrane-spanning domains. The NBDs are molecular engines, and the crystal structure of one -- the NBD from theSalmonella typhimuriumhistidine permease -- now gives us clues about how the NBDs work. The results have implications for other ABC transporters, including the protein that is mutated in cystic fibrosis.

Calorimetrically-derived parameters for protein interactions with urea and guanidine-HCl are not consistent with denaturant m values.

A recent study used calorimetric data and a stoichiometric binding model to derive binding constants, enthalpies, and stoichiometries describing the interaction between proteins and the chemical denaturants, urea and guanidine-HCl (Makhatadze and Privalov, J. Mol. Biol., 226 (1992) 491). In the present study, these parameters have been used to calculate the excess free energy, delta Gex, associated with interactions between chemical denaturants and the three proteins examined in the calorimetric study: ribonuclease A, cytochrome c, and lysozyme. This free energy and its dependence on denaturant concentration, the denaturant m value, have then been compared to experimental results from chemical denaturation experiments. The magnitudes of m values calculated from the calorimetric studies are significantly greater, 20 to 100%, than the observed values in urea. Calculated m values for guanidine-HCl range from about 10% greater than observed values for cytochrome c to over 100% greater for lysozyme. Discrepancies between calculated and observed m values are probably attributable to incomplete binding isotherms in the calorimetric studies. An additional issue raised in this study concerns the correlation of m values with changes in accessible surface areas upon unfolding. For proteins that undergo a two-state unfolding reaction, experimental m values can vary by more than a factor of two for a given protein, depending on the solution conditions. This observation suggests that factors beyond changes in accessible surface areas play a major role in determining m values.