Creation of Cross-Linked Crystals With Intermolecular Disulfide Bonds Connecting Symmetry-Related Molecules Allows Retention of Tertiary Structure in Different Solvent Conditions.

Protein crystals are generally fragile and sensitive to subtle changes such as pH, ionic strength, and/or temperature in their crystallization mother liquor. Here, using T4 phage lysozyme as a model protein, the three-dimensional rigidification of protein crystals was conducted by introducing disulfide cross-links between neighboring molecules in the crystal. The effect of cross-linking on the stability of the crystals was evaluated by microscopic observation and X-ray diffraction.

Variable and Conserved Regions of Secondary Structure in the β-Trefoil Fold: Structure Versus Function.

β-trefoil proteins exhibit an approximate C rotational symmetry. An analysis of the secondary structure for members of this diverse superfamily of proteins indicates that it is comprised of remarkably conserved β-strands and highly-divergent turn regions. A fundamental "minimal" architecture can be identified that is devoid of heterogenous and extended turn regions, and is conserved among all family members.

Functionalization of a symmetric protein scaffold: Redundant folding nuclei and alternative oligomeric folding pathways.

Successful de novo protein design ideally targets specific folding kinetics, stability thermodynamics, and biochemical functionality, and the simultaneous achievement of all these criteria in a single step design is challenging. Protein design is potentially simplified by separating the problem into two steps: (a) an initial design of a protein "scaffold" having appropriate folding kinetics and stability thermodynamics, followed by (b) appropriate functional mutation-possibly involving insertion of a peptide functional "cassette." This stepwise approach can also separate the orthogonal effects of the "stability/function" and "foldability/function" tradeoffs commonly observed in protein design.

Regenerative responses of rabbit corneal endothelial cells to stimulation by fibroblast growth factor 1 (FGF1) derivatives, TTHX1001 and TTHX1114.

Utilising rabbit corneal endothelial cells (CEC) in three different paradigms, two human FGF1 derivatives (TTHX1001 and TTHX1114), engineered to exhibit greater stability, were tested as proliferative agents. Primary CECs and mouse NIH 3T3 cells treated with the two FGF1 derivatives showed equivalent EC ranges (3.3-24 vs.

The ubiquitous buried water in the beta-trefoil architecture contributes to the folding nucleus and ~20% of the folding enthalpy.

The beta-trefoil protein architecture is characterized by three repeating "trefoil" motifs related by rotational symmetry and postulated to have evolved via gene duplication and fusion events. Despite this apparent structural symmetry, the primary and secondary structural elements typically exhibit pronounced asymmetric features. A survey of this family of proteins has revealed that among the most conserved symmetric structural elements is a ubiquitous buried solvent which participates in a bridging H-bond with three different beta-strands in each of the trefoil motifs.

Cooperative hydrophobic core interactions in the β-trefoil architecture.

Symmetric protein architectures have a compelling aesthetic that suggests a plausible evolutionary process (i.e., gene duplication/fusion) yielding complex architecture from a simpler structural motif.

Beyond the mask: a multidisciplinary reflection on palliating patients with COVID-19 receiving continuous positive airway pressure ventilation.

This narrative describes the experiences of an inner city respiratory unit that was transformed to treat COVID-19 patients with continuous positive airway pressure (CPAP) ventilation who were not scheduled for any further escalation in treatment. The high mortality rate and unconventional way of dying led to the creation of local guidance for removing assisted ventilation when the treatment ceased to be effective. We reflect on the specific challenges that caring for these patients holistically has presented and how we have learnt to deliver good palliative care in a unique set of circumstances.

Conserved buried water molecules enable the β-trefoil architecture.

Available high-resolution crystal structures for the family of β-trefoil proteins in the structural databank were queried for buried waters. Such waters were classified as either: (a) unique to a particular domain, family, or superfamily or (b) conserved among all β-trefoil folds. Three buried waters conserved among all β-trefoil folds were identified.

Oligomerization of a symmetric β-trefoil protein in response to folding nucleus perturbation.

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN).

Fine-Sampled Photographic Quantitation of Dermal Wound Healing Senescence in Aged BALB/cByJ Mice and Therapeutic Intervention with Fibroblast Growth Factor-1.

To determine quantitative parameters of dermal wound healing senescence in aged BALB/cByJ mice (an important animal model of aging) and to evaluate the potential for therapeutic intervention by fibroblast growth factor-1 (FGF-1). Utilize a novel noninvasive fine-sampled photographic methodology to quantify wound healing parameters for healing phases from wounding through to wound closure. Parameters associated with key healing phases were quantified and compared between nonaged and aged cohorts of both genders.

Crystal structure of glycosyltrehalose synthase from Sulfolobus shibatae DSM5389.

Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α-1,4 bond to an α-1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeon Sulfolobus shibatae DSM5389 (5389-GTSase) has been determined to 2.4 Å resolution by X-ray crystallography.

A mathematical model for the determination of mouse excisional wound healing parameters from photographic data.

We present a mathematical model to quantify parameters of mouse excisional wound healing from photographic data. The equation is a piecewise linear function in log scale that includes key parameters of initial wound radius (R ), an initial wound stasis phase (T ), and time to wound closure (T ); subsequently, these terms permit calculation of a latter active proliferative phase (T ), and the healing rate (HR) during this active phase. A daily photographic record of wound healing (utilizing 6 mm diameter splinted excisional wounds) permits the necessary sampling for robust parameter refinement.

Investigating the dynamics and polyanion binding sites of fibroblast growth factor-1 using hydrogen-deuterium exchange mass spectrometry.

In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR.

An S116R Phosphorylation Site Mutation in Human Fibroblast Growth Factor-1 Differentially Affects Mitogenic and Glucose-Lowering Activities.

Fibroblast growth factor-1 (FGF-1), a potent human mitogen and insulin sensitizer, signals through both tyrosine kinase receptor-mediated autocrine/paracrine pathways as well as a nuclear intracrine pathway. Phosphorylation of FGF-1 at serine 116 (S116) has been proposed to regulate intracrine signaling. Position S116 is located within a ∼17 amino acid C-terminal loop that contains a rich set of functional determinants including heparin∖heparan sulfate affinity, thiol reactivity, nuclear localization, pharmacokinetics, functional half-life, nuclear ligand affinity, stability, and structural dynamics.

Bisboronic Acids for Selective, Physiologically Relevant Direct Glucose Sensing with Surface-Enhanced Raman Spectroscopy.

This paper demonstrates the direct sensing of glucose at physiologically relevant concentrations with surface-enhanced Raman spectroscopy (SERS) on gold film-over-nanosphere (AuFON) substrates functionalized with bisboronic acid receptors. The combination of selectivity in the bisboronic acid receptor and spectral resolution in the SERS data allow the sensors to resolve glucose in high backgrounds of fructose and, in combination with multivariate statistical analysis, detect glucose accurately in the 1-10 mM range. Computational modeling supports assignments of the normal modes and vibrational frequencies for the monoboronic acid base of our bisboronic acids, glucose and fructose.

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody.

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.

Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "Second Generation" Therapeutic Application.

Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.

Evolution of a protein folding nucleus.

The folding nucleus (FN) is a cryptic element within protein primary structure that enables an efficient folding pathway and is the postulated heritable element in the evolution of protein architecture; however, almost nothing is known regarding how the FN structurally changes as complex protein architecture evolves from simpler peptide motifs. We report characterization of the FN of a designed purely symmetric β-trefoil protein by ϕ-value analysis. We compare the structure and folding properties of key foldable intermediates along the evolutionary trajectory of the β-trefoil.