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A sequence variant of human MIP-1±, in which Asp26 has been replaced by Ala, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-1± D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1² and RANTES. [ABSTRACT FROM PUBLISHER]

The article profiles the late Edwin P. Trueblood, former faculty of Earlham College, a Quaker liberal arts college in Indiana. He was born on May 16, 1861 and died on April 5, 1951 in Richmond. He served in the college from 1888 to 1939 as a teacher of speech and director of athletics. He was the youngest of nine children and graduated at Earlham in 1885 with a Bachelor of Science degree. His legacy in speech education at Earlham is discussed.

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Fragment merging is a promising approach to progressing fragments directly to on-scale potency: each designed compound incorporates the structural motifs of overlapping fragments in a way that ensures compounds recapitulate multiple high-quality interactions. Searching commercial catalogues provides one useful way to quickly and cheaply identify such merges and circumvents the challenge of synthetic accessibility, provided they can be readily identified. Here, we demonstrate that the Fragment Network, a graph database that provides a novel way to explore the chemical space surrounding fragment hits, is well-suited to this challenge. We use an iteration of the database containing >120 million catalogue compounds to find fragment merges for four crystallographic screening campaigns and contrast the results with a traditional fingerprint-based similarity search. The two approaches identify complementary sets of merges that recapitulate the observed fragment-protein interactions but lie in different regions of chemical space. We further show our methodology is an effective route to achieving on-scale potency by retrospective analyses for two different targets; in analyses of public COVID Moonshot and Mycobacterium tuberculosis EthR inhibitors, potential inhibitors with micromolar IC 50 values were identified. This work demonstrates the use of the Fragment Network to increase the yield of fragment merges beyond that of a classical catalogue search.

We present several workflows for protein-ligand docking and free energy calculation for use in the workflow management system Galaxy. The workflows are composed of several widely used open-source tools, including rDock and GROMACS, and can be executed on public infrastructure using either Galaxy's graphical interface or the command line. We demonstrate the utility of the workflows by running a high-throughput virtual screening of around 50000 compounds against the SARS-CoV-2 main protease, a system which has been the subject of intense study in the last year., (© 2022. The Author(s).)

A sequence variant of human MIP-1alpha, in which Asp26 has been replaced by Al alpha, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-alpha D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1beta and RANTES.

Background: The thrombolytic serine protease tissue-type plasminogen activator (t-PA) is a classical modular protein consisting of three types of domain in addition to the serine protease domain: F1 (homologous to fibronectin type I); G (epidermal growth factor-like) and kringle. Biochemical data suggest that the F1 and G modules play a major role in the binding of t-PA to fibrin and to receptors on hepatocytes., Results: We have derived the solution structure of the F1 and G pair of modules from t-PA by two- and three-dimensional NMR techniques, in combination with dynamical simulated annealing calculations. We have also obtained information about the molecule's backbone dynamics through measurement of amide 15N relaxation parameters., Conclusions: Although the F1 and G modules each adopt their expected tertiary structure, the modules interact intimately to bury a hydrophobic core, and the inter-module linker makes up the third strand of the G module's major beta-sheet. The new structural results allow the interpretation of earlier mutational data relevant to fibrin-binding and hepatocyte-receptor binding.

Soluble fragments of the extracellular region of vascular cell adhesion molecule 1 (VCAM-1) expressed in Escherichia coli retain functional adhesive activity. An integrin (VLA-4) binding fragment consisting of the N-terminal two immunoglobulin-like domains (VCAM-d1,2) has been crystallized. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions of a = 52.7 A, b = 66.5 A, c = 113.2 A and contain two molecules in the crystallographic asymmetric unit. A batch of protein produced in the standard E. coli strain (HW1110), but grown in the presence of selenomethionine enriched media, showed 85% incorporation of selenium in place of sulphur at methionine residues. The selenomethionyl VCAM-d1,2 was crystallized by microseeding techniques initially using the native crystals for nucleation. Both native and selenomethionyl crystals diffract X-rays to a minimum Bragg spacing of 1.8 A.

We have used an Escherichia coli expression system to produce forms of vascular cell-adhesion molecule-1 (VCAM-1) containing the first two and three supposed immunoglobulin-like domains. A form consisting of the first two domains of VCAM-1 is shown to promote very-late antigen-4-dependent spreading of a melanoma cell line comparable to that found for the equivalent region in the full seven-domain form. Preliminary structural analysis by CD and NMR is consistent with an immunoglobulin fold which is predicted from sequence comparison studies.

A segment of human tissue-type plasminogen activator (t-PA) corresponding to the fibronectin type 1 (F1) and epidermal growth factor-like (G) pair of modules, residues 1-91, has been produced as a recombinant protein in Saccharomyces cerevisiae, with a single conservative Cys to Ser substitution. The sequence-specific assignment of the 1H and 15N nuclear magnetic resonances from the pair of modules has been completed using 2D 1H nuclear magnetic resonance (NMR) spectra in conjunction with 3D, 15N-edited, 1H and 2D 15N-1H NMR spectra. Slowly exchanging amide protons have been identified, and estimates of a number of backbone 3JNH-C alpha H coupling constants were obtained by line-shape-fitting. The secondary structure of the F1 module in the pair closely matches that previously determined for the isolated F1 module from t-PA, and that of the G module conforms to the "consensus" G module structure determined previously from several isolated G modules. In the module pair, the residues linking the two modules appear to form an extended beta-strand, the carboxy-terminal end of which makes up a third strand of the major beta-sheet of the G module. The intermodule interface is defined by NOEs between residues in the ranges 22-24 in the F1 module and 65-72 in the G module. The NMR data indicate that there is little or no reorientation of the two modules with respect to one another but rather that they combine with a fixed hydrophobic contact dominated by the side chain of leucine-22.

The time between a person presenting to a general practitioner with a symptom of cancer and that person starting treatment has been studied in Devon. Retrospective analysis was undertaken of the general practitioner records of 1465 patients proven to have cancer who were registered with 245 general practitioners. During inspection of these records dates of first presentation, of referral, of first hospital consultation and of the start of treatment were noted for people with six common types of cancer (cancer of the breast, large bowel, lung, oesophagus, prostate and stomach). The general practitioner stage time and hospital stage time (pre-appointment and post-appointment) were calculated for each patient. Large differences were found in median times for the general practitioner stage according to the type of cancer, ranging from a median value of 0 days for people with breast cancer to 84 days for people with cancer of the oesophagus. For patients with cancer of the breast, large bowel, lung or prostate, median general practitioner times were shorter than median hospital stage times, while for patients with cancer of the oesophagus and stomach cancer, median general practitioner stage times were longer than median hospital stage times. Comparison of the hospital stage times for people with breast cancer and cancer of the large bowel showed notable differences between the four health districts in Devon, pre- and post-appointment times being twice as long in one district as in another. This retrospective record analysis was acceptable to participating practitioners. The results provide a basis for general practitioners and hospital staff to review their own work.

The amino acid sequence of the first domain of tissue-type plasminogen activator (t-PA) includes eight residues that are highly conserved in the type 1 finger domains found in human fibronectin. A construct comprising 50 residues from this finger domain of t-PA has been expressed and its solution structure has been determined by two-dimensional nuclear magnetic resonance spectroscopy. A total of 782 experimental restraints consisting of 723 interproton distances derived from nuclear Overhauser effect measurements, 43 torsion angles, and 16 hydrogen bond restraints were used as the input for dynamical simulated annealing structure calculations. Twenty-eight structures were obtained that satisfied the experimental data with no single distance violation greater than 0.3 A. The average atomic root-mean-square distribution for the backbone atoms of the final structures was 0.41 (+/- 0.13) A for the well defined part of the structure (residues 4 to 47). The overall fold of the t-PA finger domain shows a striking similarity to that of the seventh type 1 repeat of human fibronectin with the side-chains of conserved residues lying in similar conformations. One significant difference between the two molecules is that hydrophobic residues cover the exposed surface of the principal beta-sheet region in the t-PA finger domain. It is suggested that one face of this region may interact with parts of the complete t-PA protein.

In order to elucidate the mechanism of interaction between human epidermal growth factor (EGF) and its receptor, selected variants of EGF, differing by single amino acid substitutions, have been made by site-directed mutagenesis. The receptor affinity of these mutants was determined by a receptor binding competition assay, and the effects of the substitution on the structure of the protein were assessed by 1H nuclear magnetic resonance techniques. Various substitutions of Arg-41 resulted in substantial reduction in receptor affinity of EGF whereas change of Tyr-13 did not affect binding to the receptor. The 1H resonances of all nonexchangeable protons of the Tyr-13----Leu, Arg-41----His, and Leu-47----Glu variants were assigned and compared in order to assess the structural integrity of these mutants, which possess very different spectral and biological properties. In the case of the Leu-47----Glu mutant, only minor localized spectral changes were observed, confirming that the tertiary structure of the protein is preserved upon mutation. In contrast, for both the Arg-41----His and Tyr-13----Leu variants, significant and strikingly similar spectra changes were observed for many residues located far away from the mutated residues. This implies that similar structural alterations have taken place in both proteins, an idea further supported by hydrogen-exchange experiments where the exchange rates of hydrogen-bonded amide protons for both the Tyr-13----Leu and the Arg-41----His mutants were found to be about 4 times faster than in the wild-type protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The solution structures of the homologous growth factors human epidermal growth factor (hEGF) and human transforming growth factor-alpha (hTGF-alpha), as determined by high resolution NMR and various computational methods, are described. Knowledge of these structures and the sequences of other homologous proteins leads to predictions about growth factor residues which may be involved in the receptor/ligand interface. Recent experiments designed to check these predictions are described briefly. These involve site-specific mutagenesis, receptor binding assays and high resolution NMR studies.

The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.