13C CP-MAS and DP-MAS spin-counting experiments have been carried out on an absolute basis for a specific whole soil and its humin, humic acid, and fulvic acid fractions, as well as a sample of the soil that was treated with 2% HF(aq). The results confirm previous conclusions that a substantial fraction of the carbon content indicated by classic elemental analysis is missed in some samples, especially whole soil and humin, by both CP-MAS and DP-MAS 13C NMR methods, and that the problem is more serious for CP-MAS than for DP-MAS. This study also confirms the fact that treatment of soil organic matter with 2% HF(aq) dramatically reduces this problem but may generate some structural uncertainties associated with significant structural alterations that accompany the HF(aq) treatment, as indicated by the 13C NMR data. The relationship between the "missing carbon" problem and the concentration of paramagnetic centers, especially Fe(III) centers, is explored in substantial detail.

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Weak and transient protein–protein interactions underlie numerous biological processes. However, the location of the interaction sites of the specific complexes and the effect of transient, nonspecific protein–protein interactions often remain elusive. We have investigated the weak self-association of human growth hormone (hGH, KD = 0.90 ± 0.03 mM) at neutral pH by the paramagnetic relaxation enhancement (PRE) of the amide protons induced by the soluble paramagnetic relaxation agent, gadodiamide (Gd(DTPA-BMA)). Primarily, it was found that the PREs are in agreement with the general Hwang-Freed model for relaxation by translational diffusion (J. Chem. Phys.1975, 63, 4017–4025), only if crowding effects on the diffusion in the protein solution are taken into account. Second, by measuring the PREs of the amide protons at increasing hGH concentrations and a constant concentration of the relaxation agent, it is shown that a distinction can be made between residues that are affected only by transient, nonspecific protein–protein interactions and residues that are involved in specific protein–protein associations. Thus, the PREs of the former residues increase linearly with the hGH concentration in the entire concentration range because of a reduction of the diffusion caused by the transient, nonspecific protein–protein interactions, while the PREs of the latter residues increase only at the lower hGH concentrations but decrease at the higher concentrations because of specific protein–protein associations that impede the access of gadodiamide to the residues of the interaction surface. Finally, it is found that the ultraweak aggregation of hGH involves several interaction sites that are located in patches covering a large part of the protein surface.

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL⅐receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL⅐receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solventfilled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.Human prolactin (hPRL) is a 23-kDa protein hormone secreted both by the mammalian pituitary and numerous extrapituitary tissues. Based on similarity in quaternary structure and that of cognate receptors, prolactin is classified as a member of the larger family of hematopoietic cytokines, with highest homology to growth hormone (GH) and placental lactogen (1). Evidence suggests that hPRL functions as an autocrine-paracrine growth factor in cancers of the breast, prostate, and female reproductive tract. Both hPRL and its receptor (hPRLr) are expressed in a majority of breast cancers (2, 3), where their interaction affects various aspects of carcinogenesis (2-5). Cellular studies of hPRL signaling provide a molecular basis for the hPRL role in breast cancer proliferation (6 -10). Recent epidemiological studies have strengthened the link between elevated hPRL and breast cancer risk (10). Consequently, detailed molecular understanding of hPRL⅐receptor recognition and activation is critical to aid efforts to develop potential cancer intervention strategies.hPRL recognizes the hPRLr extracellular domain (ECD) and recruits two molecules of hPRLr via two distinct binding sites on the hPRL surface, i.e. site 1 (high affinity) followed by site 2 (low affinity) (11). Crystallographic structures of 1:1 (12) (hPRL antagonist⅐hPRLrECD) and 1:2 (13) (so called "affinity matured" hPRL⅐hPRLrECD) complexes reveal molecular details of the ϳ20% of the combined surface area buried within the high affinity binding interface. Cumulative work with mutagenic variants of hPRL further reduces the number of critical residue contacts for receptor binding (14 -16). Associated with formation of the heterotrimeric 1:2 (ligand⅐receptor) complex, induced conformational changes in the receptor intracellular domain brings two receptor-associated cytosolic tyrosine kinase molecules of Janus kinase-2 (JAK2)...