Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K

The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and Rfree = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6–Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S–S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported

A Comprehensive Resource of Interacting Protein Regions for Refining Human Transcription Factor Networks

Large-scale data sets of protein-protein interactions (PPIs) are a valuable resource for mapping and analysis of the topological and dynamic features of interactome networks. The currently available large-scale PPI data sets only contain information on interaction partners. The data presented in this study also include the sequences involved in the interactions (i.e., the interacting regions, IRs) suggested to correspond to functional and structural domains. Here we present the first large-scale IR data set obtained using mRNA display for 50 human transcription factors (TFs), including 12 transcription-related proteins. The core data set (966 IRs; 943 PPIs) displays a verification rate of 70%. Analysis of the IR data set revealed the existence of IRs that interact with multiple partners. Furthermore, these IRs were preferentially associated with intrinsic disorder. This finding supports the hypothesis that intrinsically disordered regions play a major role in the dynamics and diversity of TF networks through their ability to structurally adapt to and bind with multiple partners. Accordingly, this domain-based interaction resource represents an important step in refining protein interactions and networks at the domain level and in associating network analysis with biological structure and function

Determining Stable Single Alpha Helical (SAH) Domain Properties by Circular Dichroism and Atomic Force Microscopy

Stable, single α-helical (SAH) domains exist in a number of unconventional myosin isoforms, as well as other proteins. These domains are formed from sequences rich in charged residues (Arg, Lys, and Glu), they can be hundreds of residues long, and in isolation they can tolerate significant changes in pH and salt concentration without loss in helicity. Here we describe methods for the preparation and purification of SAH domains and SAH domain-containing constructs, using the myosin 10 SAH domain as an example. We go on to describe the use of circular dichroism spectroscopy and force spectroscopy with the atomic force microscope for the elucidation of structural and mechanical properties of these unusual helical species

The recycling of carbon in glucose, lactate and alanine in sheep

Pregnant ewes with catheters implanted in an artery and the uterine and recurrent tarsal veins were infused at a constant rate with U−¹⁴C-labelled glucose, alanine or bicarbonate. Measurements were made of the overall and local fractional contribution of glucose and alanine to CO₂ production and of the extent of interconversion of these metabolites. In the whole animal, by coupling the results with the authors’ previous study of lactate metabolism, a solution was obtained to an open unrestricted 4-compartment model of the exchange of carbon between glucose, lactate, alanine and CO₂. A more limited study was made with non-pregnant sheep because complete data for lactate interactions with alanine were not available. Our analysis of glucose/lactate/alanine/CO₂ interactions in pregnant sheep suggests that about two-thirds of the glycogenic carbon was oxidised fairly directly to CO₂. There was relatively little recycling of glucose carbon through lactate and alanine so that most of the remaining glycogenic carbon was stored as product with relatively long turnover time. It is possible that much of this was in the form of muscle glycogen, and analysis of glycogenic carbon exchange across the hind limb muscle was consistent with this conclusion. In non-pregnant ewes, the findings, although incomplete, suggested that there were no great differences from the findings in pregnant ewes.Derek B. Lindsay, Patrick J. Barker, Andrew J. Northrop, Brian P. Setchell, Graham J. Faichne