DSC study of sucrose melting

An early endothermic peak at approximately 150 degrees C was observed for crystalline sucrose by differential scanning calorimetry. The enthalpy at this temperature was found to vary with recrystallised sucrose from different sources. The addition of mineral salts to recrystallisation solutions decreased the enthalpy of the peak at around 150 degrees C, whereas the absence of salts increased it. The presence of organic solvents and polysaccharides in solution had a minor effect compared to the inorganic impurities. The peak was also depleted by increasing the amount of stirring and temperature at which recrystallisation was performed.     

NMR investigation of DNA primer-template models: structural insights into dislocation mutagenesis in DNA replication

Slipped frameshift intermediates can occur when DNA polymerase slows or stalls at sites of DNA lesions. However, this phenomenon is much less common when unmodified DNA is replicated. In order to study the effect of templating bases on the alignment of primer-templates, NMR structural investigation has been performed on primer-template oligonucleotide models which mimic the situation that dNTP has just been incorporated opposite template. NMR evidence reveals the occurrence of misalignment when dGTP is incorporated opposite template T with a downstream nucleotide C. Depending on the template sequence, further extension of the primer can lead to realignment.     

Thermodynamic analysis of water molecules at the surface of proteins and applications to binding site prediction and characterization

Water plays an essential role in determining the structure and function of all biological systems. Recent methodological advances allow for an accurate and efficient estimation of the thermodynamic properties of water molecules at the surface of proteins. In this work, we characterize these thermodynamic properties and relate them to various structural and functional characteristics of the protein. We find that high-energy hydration sites often exist near protein motifs typically characterized as hydrophilic, such as backbone amide groups. We also find that waters around alpha helices and beta sheets tend to be less stable than waters around loops. Furthermore, we find no significant correlation between the hydration site-free energy and the solvent accessible surface area of the site. In addition, we find that the distribution of high-energy hydration sites on the protein surface can be used to identify the location of binding sites and that binding sites of druggable targets tend to have a greater density of thermodynamically unstable hydration sites. Using this information, we characterize the FKBP12 protein and show good agreement between fragment screening hit rates from NMR spectroscopy and hydration site energetics. Finally, we show that water molecules observed in crystal structures are less stable on average than bulk water as a consequence of the high degree of spatial localization, thereby resulting in a significant loss in entropy. These findings should help to better understand the characteristics of waters at the surface of proteins and are expected to lead to insights that can guide structure-based drug design efforts.     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

A survey of the year 2006 literature on applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is a fast and robust method to determine the energetics of association reactions in solution. The changes in enthalpy, entropy and heat capacity that accompany binding provide unique insights into the balance of forces driving association of molecular entities. ITC is used nowadays on a day-to-day basis in hundreds of laboratories. The method aids projects both in basic and practice-oriented research ranging from medicine and biochemistry to physical chemistry and material sciences. Not surprisingly, the range of studies utilizing ITC data is steadily expanding. In this review, we discuss selected results and ideas that have accumulated in the course of the year 2006, the focus being on biologically relevant systems. Theoretical developments, novel applications and studies that provide a deeper level of understanding of the energetic principles of biological function are primarily considered. Following the appearance of a new generation of titration calorimeters, recent papers provide instructive examples of the synergy between energetic and structural approaches in biomedical and biotechnological research.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Protein heat capacity reflects the dynamics of enthalpy exchange between the single macromolecule and the surroundings

Heat capacity has played a prominent role in relating macroscopic and microscopic properties of small molecules and crystals. However, its diagnostic power can also be used for macromolecules such as proteins. It is shown in the present study that the macroscopically observed protein heat capacity provides direct access to the thermodynamic state of the single protein molecule. The new model of the physical basis of protein heat capacity emphasizes the dynamic nature of protein molecules. It incorporates equilibrium fluctuations as an integral constituent and shows that the increase in the magnitude of equilibrium fluctuations is coupled to an increase in the enthalpy flux between the individual protein molecule and its surroundings. Proteins 2000;41:86–92. © 2000 Wiley‐Liss, Inc.     

Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride

Enthalpy and heat capacity changes for the deprotonation of 18 buffers were calorimetrically determined in 0.1 M potassium chloride at temperatures ranging from 5 to 45°C. The values of the dissociation constant were also determined by means of potentiometric titration. The enthalpy changes for the deprotonation of buffers, except for the phosphate and glycerol 2-phosphate buffers, were found to be characterized by a linear function of temperature. The enthalpy changes for the second dissociation of phosphate and glycerol 2-phosphate where divalent anion is formed on dissociation were fitted with the second order function of temperature rather than the first order. Temperature dependence of buffer pH calculated by using the enthalpy and heat capacity changes obtained was in good agreement with the temperature variation of the pH values actually measured in the temperature range between 0 and 50°C for all the buffers studied. On the basis of the results obtained, a numeric table showing the temperature dependence of pK values for the 18 buffers is presented. Proteins 33:159–166, 1998. © 1998 Wiley-Liss, Inc.     

Local control of peptide conformation: Stabilization of cis proline peptide bonds by aromatic proline interactions

In the native state of proteins there is a marked tendency for an aromatic amino acid to precede a cis proline. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. In order to study the role that local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence acetyl-Gly-X-Pro-Gly-carboxamide (GXPG), where X = Tyr, Phe, Trp, Ala, or cyclohexyl alanine, were synthesized and studied by nmr. Analysis of the nmr data shows that none of the peptides adopt a specific backbone structure. Ring current shifts, the equilibrium constants, the Van't Hoff enthalpy, and the measured rate of cis-trans isomerization all indicate that the cis proline conformer is stabilized by favorable interactions between the aromatic ring and the proline residue. Analysis of the side chain conformation of the aromatic residue and analysis of the chemical shifts of the pyrrolidine ring protons shows that the aromatic side chain adopts a preferred conformation in the cis form. The distribution of rotamers and the effect of an aromatic residue on the cis-trans equilibrium indicate that the preferred conformation is populated to approximately 62% for the Phe containing peptide, 67% for the Tyr containing peptide, and between 75 and 80% for the Trp containing peptide. The interaction is unaffected by the addition of 8M urea. These local interactions favor an aromatic residue immediately preceding a cis proline, but they cannot explain the relative propensities for Phe-Pro, Tyr-Pro, and Trp-Pro cis peptide bonds observed in the native state of proteins. In the model peptides the percentage of the cis proline conformer is 21% GYPG while it is 17% for GFPG. This difference is considerably smaller than the almost three to one preponderance observed for cis Tyr-Pro peptide bonds vs cis Phe-Pro peptide bonds in the protein database. © 1998 John Wiley & Sons, Inc. Biopoly 45: 381–394, 1998     

Mechanistic aspects of chiral discrimination by surface‐immobilized α1‐acid glycoprotein

The immobilization of the globular protein α‐1‐acid glycoprotein (AGP) onto silica gel led to the commercial availability of an AGP column, which has a high enantioselectivity. The enantioselectivity of AGP columns has been demonstrated in numerous applications. Due to potential AGP structural changes occurring upon its immobilization, the interaction between particular pairs of enantiomers and the stationary phase is very difficult to assess. Therefore, in this paper we report a mechanistic study that probes the nature of these types of interactions. As model ligands, we employed two LTD₄ antagonists (L‐708, 738, MK0476, and their enantiomers) which have a rigid backbone consisting of a conjugated aromatic region and a side chain which is terminated with a carboxylic functional group. The difference between the two compounds is a two‐fluorine versus one‐chlorine substituent in the aromatic region of the molecule. To study the interaction between the two homologues and the AGP stationary phase, several parameters were varied, including pH, ionic strength, organic modifier, and temperature. van't Hoff plots were constructed and found to be nonlinear. They could, however, be divided into two linear regions, one from 0°C to ∼30°C, and another from 39°C to 50°C. The region at lower temperature implied that the separation was entropy‐dominated while the separation at higher temperature was enthalpically driven. The transition from the entropic to the enthalpically driven separation region suggested that bound AGP undergoes a conformational change. Fluorescence spectroscopy performed on the AGP stationary phase found evidence for a limited conformational transition at a similar temperature, consistent with this hypothesis. Chirality 11:224–232, 1999. © 1999 Wiley‐Liss, Inc.