A proteome-wide quantitative platform for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs.

The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access quantitative database that provides the most efficient extraction conditions of 2065 unique mammalian MPs. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to capture membrane 'nano-scoops' containing a target MP efficiently and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

Nanostructured lipopeptide-based membranomimetics for stabilizing bacteriorhodopsin.

Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein, bacteriorhodopsin. Formation of a cylindrical-type micelle assembly induced by facial amphipathic lipopeptides resembles a biological membrane more effectively than conventional micelles. The hydrophobic face of this cylindrical-type micelle provides extended stability to the membrane protein and the hydrophilic surface interacts with an aqueous environment. In our present study, we have demonstrated experimentally and computationally that lipopeptide-based facial detergents having an unstructured or ß-turn conformation can stabilize membrane proteins. However, constrained peptide detergents can provide enhanced stability to bacteriorhodopsin. In this study, we have computationally examined the structural stability of bacteriorhodopsin in the presence of helical, beta-strand, and cyclic unstructured peptide detergents, and conventional detergent-like peptides. Our study demonstrates that optimal membranomimetics (detergents) for stabilizing a specific membrane protein can be screened based on the following criteria: (i) hydrodynamic radii of the self-assembled peptide detergents, (ii) stability assay of detergent-encased membrane proteins, (iii) percentage covered area of detergent-encased membrane proteins obtained computationally and (iv) protein-detergent interaction energy.

Engineered Bio-inspired Multifunctional Peptide- and Protein-based Therapeutic Biomolecules for Better Wound Care.

Developing non-immunogenic therapeutic biomolecules for facilitating blood clotting followed by wound healing via therapeutic angiogenesis, still remains a formidable challenge. Excessive blood loss of accident victims and battalions cause a huge number of deaths worldwide. Patients with inherited bleeding disorders face acute complications during injury and post-surgery. Biologically-inspired peptide-based hemostat can act as a potential therapeutic for handling coagulopathy. Additionally, non-healing wounds for patients having ischemic diseases can cause severe clinical complications. Advancement in stabilized growth-factor-based proangiogenic therapy may offer effective possibilities for the treatment of ischemic pathology. This review will discuss nature-inspired biocompatible stabilized peptide- and protein-based molecular medicines to serve unmet medical challenges for handling traumatic coagulopathy and impaired wound healing.

Peptide-based topical agents and intravenous hemostat for rapid hemostasis.

Traumatic coagulopathy due to severe external injury and internal hemorrhage is life-threatening to accident victims and soldiers on the battlefield, causing considerable number of deaths worldwide. Patients with inherited bleeding disorders (such as haemophilia, von Willebrand disease, inherited qualitative platelet defects, and afibrinogenemia) also contribute to the vast number of deaths due to abnormal bleeding, and these patients are difficult to handle during surgery. Platelets and different plasma proteins play an essential role in blood coagulation and in the maintenance of the body's hemostatic balance. The improper function or deficiency of these factors cause abnormal bleeding. To address such bleeding disorders, external clotting agents (such as extracellular protein-inspired natural and synthetic peptide-based sealants and peptide-functionalized polymer/liposome-based sealants) have been developed by different groups of researchers. The primary focus of this review is to provide molecular insights into the existing biologically inspired peptide-based sealants, highlighting the advantages and limitations of such reported designed sealants to handle blood clotting, and also provide insights into the design of improved next-generation surgical sealants.

Bioinspired Non-Immunogenic Multifunctional Sealant for Efficient Blood Clotting and Suture-Free Wound Closure.

Engineering bioinspired peptide-based molecular medicine is an emerging paradigm for the management of traumatic coagulopathies and inherent bleeding disorder. A hemostat-based strategy in managing uncontrolled bleeding is limited due to the lack of adequate efficacy and clinical noncompliance. In this study, we report an engineered adhesive peptide-based hybrid regenerative medicine, sealant 5, which is designed integrating the structural and functional features of fibrin and mussel foot-pad protein. AFM studies have revealed that sealant 5 (55.8 ± 6.8 nN adhesive force) has higher adhesive force than fibrin (46.4 ± 7.3 nN adhesive force). SEM data confirms that sealant 5 retains its network-like morphology both at 37 and 60 °C, inferring its thermal stability. Both sealant 5 and fibrin exhibit biodegradability in the presence of trypsin, and sealant 5 also showed biocompatibility in the presence of fibroblast cells. Engineered sealant 5 efficiently promotes hemostasis with enhanced adhesiveness and less blood-loss than fibrin. In vivo data suggests that in heparinized conditions, sealant 5 ceases bleeding at 212.3 ± 15.1 s, whereas fibrin halts bleeding at 294.3 ± 21.4 s and blood-loss is ∼4-fold less in sealant 5 than in fibrin. In a heparinized system, sealant 5 facilitates faster blood-clotting than fibrin (∼82 s faster) and RADA-16, a reported peptide-based sealant (∼113 s faster). Additionally, in the case of sealant 5, the process of clotting mimicry-like fibrin is independent of the body's own coagulation system. Sealant 5 efficiently halts bleeding for both external and internal wounds, even for a heparinized system overcoming the bacterial infection. ELISA data and PMBC cell proliferation data support the non-immunogenic feature of sealant 5. Though fibrin and sealant 5 have exhibited comparable efficacy in suture-free wound closure, in vivo H&E staining images have revealed infiltration of very few immune cells as well as the presence of abundant collagen formation in the case of sealant 5-treated wound. Such nature-inspired non-immunogenic sealants offer exciting possibilities for the treatment of uncontrolled bleeding vis-à-vis wound closure.

Engineered isopeptide bond stabilized fibrin inspired nanoscale peptide based sealants for efficient blood clotting.

Designing biologically inspired nanoscale molecular assembly with desired functionality is a challenging endeavour. Here we report the designing of fibrin-inspired nanostructured peptide based sealants which facilitate remarkably fast entrapping of blood corpuscles (~28 seconds) in contrast to fibrin (~56 seconds). Our engineered sealants are stabilized by lysine-aspartate ionic interactions and also by Nε(γ-glutamyl) lysine isopeptide bond mediated covalent interaction. Each sealant is formed by two peptides having complementary charges to promote lysine-aspartate ionic interactions and designed isopeptide bond mediated interactions. Computational analysis reveals the isopeptide bond mediated energetically favourable peptide assemblies in sealants 1-3. Our designed sealants 2 and 3 mimic fibrin-mediated clot formation mechanism in presence of transglutaminase enzyme and blood corpuscles. These fibrin-inspired peptides assemble to form sealants having superior hemostatic activities than fibrin. Designed sealants feature mechanical properties, biocompatibility, biodegradability and high adhesive strength. Such nature-inspired robust sealants might be potentially translated into clinics for facilitating efficient blood clotting to handle traumatic coagulopathy and impaired blood clotting.

A study of piezoelectric and mechanical anisotropies of the human cornea.

The piezoelectric and dynamic mechanical properties of human cornea have been investigated as a function of drying time. As expected, the piezoelectric coefficient, d(31), and the Young's modulus, Y, were found to be extremely sensitive to water content. d(31) decreased with dehydration of the corneal tissue and Y increased with dehydration. While these results are significant, the discovery of the unprecedented mechanical and electromechanical anisotropy exhibited by the cornea are the major findings of this study and indicate that the collagen fibrils comprising the cornea are highly oriented. The piezoelectric responses of corneas observed in this study are: diagonally cut samples starting at an average piezoelectric coefficient value of 2250 pC/N, followed by the vertically cut samples, with an average starting value of about 600 pC/N and finally the horizontally cut samples with an average starting value of about 200 pC/N.