Identification of protein–protein interfaces by decreased amide proton solvent accessibility

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein–protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5–24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5–24)-binding site. A complex of unknown structure also was analyzed, human α-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4–5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity.

Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility

Little is known about stem cell biology or the specialized environments or niches believed to control stem cell renewal and differentiation in self-renewing tissues of the body. Functional assays for stem cells are available only for hematopoiesis and spermatogenesis, and the microenvironment, or niche, for hematopoiesis is relatively inaccessible, making it difficult to analyze donor stem cell colonization events in recipients. In contrast, the recently developed spermatogonial stem cell assay system allows quantitation of individual colonization events, facilitating studies of stem cells and their associated microenvironment. By using this assay system, we found a 39-fold increase in male germ-line stem cells during development from birth to adult in the mouse. However, colony size or area of spermatogenesis generated by neonate and adult stem cells, 2–3 months after transplantation into adult tubules, was similar (∼0.5 mm2). In contrast, the microenvironment in the immature pup testis was 9.4 times better than adult testis in allowing colonization events, and the area colonized per donor stem cell, whether from adult or pup, was about 4.0 times larger in recipient pups than adults. These factors facilitated the restoration of fertility by donor stem cells transplanted to infertile pups. Thus, our results demonstrate that stem cells and their niches undergo dramatic changes in the postnatal testis, and the microenvironment of the pup testis provides a more hospitable environment for transplantation of male germ-line stem cells.

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.