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Accurate prediction of location of cavities and surface grooves in proteins is important, as these are potential sites for ligand binding. Several currently available programs for cavity detection are unable to detect cavities near the surface or surface grooves. In the present study, an optimized molecular dynamics based procedure is described for detection and quantification of interior cavities as well as surface pockets. This is based on the observation that the mobility of water in such pockets is significantly lower than that of bulk water. The algorithm efficiently detects surface grooves that are sites of protein-ligand and protein-protein interaction. The algorithm was also used to substantially improve the performance of an automated docking procedure for docking monomers of nonobligate protein-protein complexes. In addition, it was applied to predict key residues involved in the binding of the E. coli toxin CcdB with its inhibitor. Predictions were subsequently validated by mutagenesis experiments.

Globular protein thermostability is characterized the cold denaturation, maximal stability $(T_{ms})$ and heat denaturation temperatures. For mesophilic globular proteins, $T_{ms}$ typically ranges from −25°C to +35°C. We show that the indirect estimate of $T_{ms}$ from calorimetry and the direct estimate from chemical denaturation performed in a range of temperatures are in close agreement. The heat capacity change of unfolding per mol residue $(\Delta C_p)$ alone is shown to accurately predict $T_{ms}.\Delta C_p$ and hence $T_{ms}$ can be predicted solely from the protein sequence. The average difference in free energy of unfolding at the observed and predicted values of $T_{ms}$ is 1.0 kcal $mol^{-1}$, which is small compared to typical values of the total free energy of unfolding.

Maltose binding protein (MBP) exhibits a slow phase of folding at pH 7.4, 298 K. The kinetics of this phase has been characterized as a function of denaturant concentration and temperature. Denaturant double-jump experiments and the activation energy for folding indicate that the slow phase involves processes other than proline isomerization. Although the first five N-terminal residues are disordered in the MBP crystal structure, mutations in this region slow down folding and destabilize the native structure. This is the first report showing that disordered N-terminal residues can affect folding kinetics and stability.

A full-length cDNA clone for human myoglobin has been isolated from a human skeletal muscle cDNA library. The clone as isolated has a cDNA insert approximately one kilobase long and has 5' and 3' untranslated regions of approximately 80 and 530 base pairs, respectively. The sequence of the translated region corresponds exactly to that predicted for human myoglobin. The cDNA was expressed in high yield in Escherichia coli as a fusion protein consisting of the first 31 amino acids of the phage lambda cII gene, the tetrapeptide Ile-Glu-Gly-Arg, and the myoglobin sequence by following the approach of Nagai and Thogersen [Nagai, K. & Thogersen, M. C. (1984) Nature (London) 309, 810-812]. The fusion product was isolated, reconstituted with heme, cleaved with trypsin, and purified to generate a protein whose properties are indistinguishable from those for authentic human myoglobin. Myoglobin can be readily prepared on a gram scale by using these methods.

Residue Val68 in human myoglobin has been replaced by Asn, Asp, and Glu with site-directed mutagenesis. Purified proteins were characterized by isoelectric focusing and by absorption, CD, and NMR spectroscopy. These studies demonstrated that Mb is able to tolerate substitution of the buried hydrophobic residue Val68 by Asn, Asp, and Glu. In the metaquo derivatives of the Glu and Asp mutants, the negative charge at residue 68 is stabilized by a favorable Coulombic interaction with the heme iron. In the absence of this interaction, as in the metcyano and ferrous deoxy derivatives, the relatively nonpolar protein interior cannot stabilize an isolated buried negative charge, and the carboxylate is either protonated or stabilized via a salt bridge with the nearby distal histidine. Hence in the Asp and Glu mutant proteins, both reduction and cyanide binding are accompanied by proton uptake by the protein. The apoproteins were prepared and reconstituted with the chlorophyll derivative zinc pyrochlorophyllide a. Absorption and fluorescence spectra were quite similar for wild-type and all mutant proteins reconstituted with this derivative. These results do not support the point charge model for the red shifts observed in the spectra of chlorophylls associated with photosynthetic proteins. From the pH dependence of the absorption spectrum of zinc pyrochlorophyllide a in the Glu mutant, the apparent pK, of the buried glutamate residue was estimated to be 8.9. This increase of 4.4 pH units, over the value for Glu in aqueous solution, provides a measure of the polarity of the protein interior.

The temperature dependences of the reduction potentials (E degrees') of wild-type human myoglobin (Mb) and three site-directed mutants have been measured by the use of thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. The changes in E degrees' and the standard entropy (delta S degrees') and enthalpy (delta H degrees') of reduction in the mutant proteins were determined relative to values for wild type; the change in E degrees' at 25 degrees C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. At pH 7.0, reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton by the protein. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein.

The S protein-S peptide interaction is a model system to study binding thermodynamics in proteins. We substituted alanine at position 4 in S peptide by alpha-aminoisobutyric acid (Aib) to investigate the effect of this substitution on the conformation of free S peptide and on its binding to S protein. The thermodynamic consequences of this replacement were studied using isothermal titration calorimetry. The structures of the free and complexed peptides were studied using circular dichroic spectroscopy and X-ray crystallography, respectively. The alanine4Aib replacement stabilizes the free S peptide helix and does not perturb the tertiary structure of RNase S. Surprisingly, and in contrast to the wild-type S peptide, the DeltaG degrees of binding of peptide to S pro, over the temperature range 5-30 degrees C, is virtually independent of temperature. At 25 degrees C, the DeltaDeltaG degrees, DeltaDeltaH degrees, DeltaDeltaS and DeltaDeltaCp of binding are 0.7 kcal/mol, 2.8 kcal/mol, 6 kcal/mol x K and -60 kcal/mol x K, respectively. The positive value of DeltaDeltaS is probably due to a decrease in the entropy of uncomplexed alanine4Aib relative to the wild-type peptide. The positive value of DeltaDeltaH: degrees is unexpected and is probably due to favorable interactions formed in uncomplexed alanine4Aib. This study addresses the thermodynamic and structural consequences of a replacement of alanine by Aib both in the unfolded and complexed states in proteins.

The rate of oxidation of formic acid by CoIIIaq has been measured in aqueous perchlorate media over a wide range of conditions using conventional and stopped-flow spectrophotometry. The order in [CoIII] is unity and the order in [HCOOH] lies between zero and unity; the rate decreases with increasing acidity in a complex manner. The mechanism is resolved into two rate determining processes, each involving an intermediate CoIII—HCOOH complex. These two complexes have an acid–base relationship involving two protons. Values for ΔH* and ΔS* together with the ratio of rates of oxidation of HCOOH and DCOOH are compared for a range of oxidizing ions.

The kinetics of the oxidation of isopropanol by CoIIIaq have been studied using conventional and stopped-flow spectrophotometry in aqueous perchlorate media at high values for the ratio (initial [isopropanol])/(initial[CoIIIaq]) where Δ[CoIIIaq]/Δ[acetone]= 2.0. The rate is first order in [CoIIIaq] and first order in [isopropanol], and there is no evidence for intermediate complexes at very high [isopropanol]. The variation of the observed second order rate constant with acidity fits a mechanism where both CoIIIaq and CoIIIOH–aq react with the unprotonated alcohol, and energies E and entropies ΔS* of activation are compared with E and ΔS* for the oxidation of isopropanol by other cations and for the oxidation of other substrates by CoIIIaq in aqueous perchlorate media. The published data for the rates of oxidation of other alcohols by CoIIIaq conform to a mechanism similar to that for isopropanol, and the rate constants for various substrates with a range of oxidants are compared.

In current seasonal influenza vaccines, neutralizing antibody titers directed against the hemagglutinin surface protein are the primary correlate of protection. These vaccines are, therefore, quantitated in terms of their hemagglutinin content. Adding other influenza surface proteins, such as neuraminidase and M2e, to current quadrivalent influenza vaccines would likely enhance vaccine efficacy. However, this would come with increased manufacturing complexity and cost. To address this issue, as a proof of principle, we have designed genetic fusions of hemagglutinin ectodomains from H3 and H1 influenza A subtypes. These recombinant H1-H3 hemagglutinin ectodomain fusions could be transiently expressed at high yield in mammalian cell culture using Expi293F suspension cells. Fusions were trimeric, and as stable in solution as their individual trimeric counterparts. Furthermore, the H1-H3 fusion constructs were antigenically intact based on their reactivity with a set of conformation-specific monoclonal antibodies. H1-H3 hemagglutinin ectodomain fusion immunogens, when formulated with the MF59 equivalent adjuvant squalene-in-water emulsion (SWE), induced H1 and H3-specific humoral immune responses equivalent to those induced with an equimolar mixture of individually expressed H1 and H3 ectodomains. Mice immunized with these ectodomain fusions were protected against challenge with heterologous H1N1 (Bel/09) and H3N2 (X-31) mouse-adapted viruses with higher neutralizing antibody titers against the H1N1 virus. Use of such ectodomain-fused immunogens would reduce the number of components in a vaccine formulation and allow for the inclusion of other protective antigens to increase influenza vaccine efficacy.

Maltose binding protein (MBP) is a 370aa protein found in the periplasmic space of gram-negative bacteria and serves as a receptor for osmotic shock sensitivity and chemotaxis in response to the maltose and other maltodextrins from the environment. X-ray crystallographic experiments have shown a ligand induced conformational change in the binding protein structure, resulting in the two N- and C- terminal clefts coming closer to each other and thus enclosing the ligand molecule in the spatially buried binding groove. Here, we are interested in capturing the effect of the ligands on the protein conformation, one molecule at a time, by using atomic force microscope. Through single- molecule experiments, we are trying to understand the effect of maltose and maltotriose on the mechanical stability and the unfolding pathways of MBP. Our results have shown that MBP unfolds via at least two different pathways: one being a two-state unfolding pathway occurring in 38% cases whereas the other contains a mechanically stable intermediate occurring in the remaining 62% cases. The main unfolding peak is observed at ∼75 pN, unraveling a contour length of 100 ± 8 nm whereas the intermediate state occurs at almost half the length of the main state, i.e. at 50 ± 8 nm and at force of ∼45 pN. Although the presence of its ligand, maltose, shows no change in the unfolding force or the contour length of both the states during unfolding, but an increase has been observed in the frequency of occurrence of the intermediate (79%). This increase is more pronounced in case of maltotriose bound protein (83%). This indicates that the ligand bound MBP prefers to unfold via an intermediate.