The passive elasticity of muscle is largely governed by the I-band part of the giant muscle protein titin, a complex molecular spring composed of a series of individually folded immunoglobulin-like domains as well as largely unstructured unique sequences. These mechanical elements have distinct mechanical properties, and when combined, they provide the desired passive elastic properties of muscle, which are a unique combination of strength, extensibility and resilience. Single-molecule atomic force microscopy (AFM) studies demonstrated that the macroscopic behaviour of titin in intact myofibrils can be reconstituted by combining the mechanical properties of these mechanical elements measured at the single-molecule level. Here we report artificial elastomeric proteins that mimic the molecular architecture of titin through the combination of well-characterized protein domains GB1 and resilin. We show that these artificial elastomeric proteins can be photochemically crosslinked and cast into solid biomaterials. These biomaterials behave as rubber-like materials showing high resilience at low strain and as shock-absorber-like materials at high strain by effectively dissipating energy. These properties are comparable to the passive elastic properties of muscles within the physiological range of sarcomere length and so these materials represent a new muscle-mimetic biomaterial. The mechanical properties of these biomaterials can be fine-tuned by adjusting the composition of the elastomeric proteins, providing the opportunity to develop biomaterials that are mimetic of different types of muscles. We anticipate that these biomaterials will find applications in tissue engineering as scaffold and matrix for artificial muscles.

atomic force microscopy ͉ protein-ligand binding ͉ protein-protein interaction P rotein-ligand interactions, including protein-protein interactions, play crucial roles in almost all biological processes and functions and have important applications in medicine and biotechnology (1). The binding of a ligand to the protein will induce conformational change of the protein, which can be a minute structural perturbation or a large conformation change, and transform the protein into a new functional state that is distinct from the ligand-free form of the protein. This new functional state then can trigger a cascade of biological reactions (2, 3). Many techniques have been developed to characterize protein-ligand interactions and measure their binding affinity in vitro and in vivo (4-6). However, most of the techniques are largely based on colocalization of the proteins and their interacting partners and involve the detection of change of physical properties upon binding of the ligand, such as fluorescence and refractive index, which are not necessarily the structural or functional consequence of ligand binding. However, the functional protein-ligand complexes (ligand-bound functional states) are not merely the colocalization of the two interacting partners. Instead, it is the structural difference, being minute or large, and its functional consequence that distinguish the functional ligand-bound form from the nonfunctional ligand-free form. Hence, it is of critical importance to probe the structural and/or functional consequence of the protein upon binding of ligands and develop functional binding assay to directly report the functional state of the protein-ligand complex.

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.Small proteins (with fewer than 100 amino acids) are still under the scrutiny of having smooth versus the rough energy landscape; however, for proteins larger than 100 amino acid residues it is generally believed that they follow multiple pathways involving one or many intermediates during protein unfolding (1-4). Contrary to this notion, there exist few exceptions among large proteins, for example, Borrelia burgdorferi VlsE (5), Alteromonas haloplanctis ␣-amylase (6), and maltosebinding protein (MBP) 2 (7), which are shown to exhibit an apparent two-state equilibrium unfolding pathway in bulk solution. Very recent single-molecule pulling studies showed that out-of-equilibrium mechanical unfolding of MBP is very complex and consists of on-pathway sequential intermediates (8, 9). Our earlier studies on MBP have shown the existence of intermediates during the folding pathway of MBP (10, 11). Traditional bulk biophysical techniques like fluorescence, time-resolved unfolding and folding probe ensembles of molecules and hence suffer many drawbacks compared with methods that probe one molecule at a time. Therefore, single-molecule studies on protein folding/unfolding have proved vital in givin...

CD4 is an important component of the immune system and is also the cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. Constructs consisting of all four extracellular domains of human CD4 as well as the first two domains (CD4D12) have previously been expressed and characterized. All of the gp120-binding residues are located within the first N-terminal domain (D1) of CD4. To date, it has not been possible to obtain domain D1 alone in a soluble and active form. Most residues in CD4 that interact with gp120 lie within the region 21-64 of domain D1 of CD4. On the basis of these observations and analysis of the crystal structure of CD4D12, a mutational strategy was designed to express CD4D1 and region 21-64 of CD4 (CD4PEP1) in Escherichia coli. K(D) values for the binding of CD4 analogues described above to gp120 were measured using a Biacore-based solution-phase competition binding assay. Measured K(D) values were 15 nM, 40 nM, and 26 microM for CD4D12, CD4D1, and CD4PEP1, respectively. All of the proteins interact with gp120 and are able to expose the 17b-binding epitope of gp120. Structural content was determined using CD and proteolysis. Both CD4D1 and CD4PEP1 were partially structured and showed an enhanced structure in the presence of the osmolyte sarcosine. The aggregation behavior of all of the proteins was characterized. While CD4D1 and CD4PEP1 did not aggregate, CD4D12 formed amyloid fibrils at neutral pH within a week at 278 K. These CD4 derivatives should be useful tools in HIV vaccine design and entry inhibition studies.

In Saccharomyces cerevisiae, the mitochondrial inner membrane readily allows transport of cytosolic NAD ؉ , but not

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In this work, we have synthesized a series of novel C,Ncyclometalated 2H-indazole-ruthenium(II) and -iridium(III) complexes with varying substituents (H, CH 3 , isopropyl, and CF 3 ) in the R 4 position of the phenyl ring of the 2H-indazole chelating ligand. All of the complexes were characterized by 1 H, 13 C, high-resolution mass spectrometry, and elemental analysis. The methyl-substituted 2H-indazole-Ir(III) complex was further characterized by single-crystal X-ray analysis. The cytotoxic activity of new ruthenium(II) and iridium(III) compounds has been evaluated in a panel of triple negative breast cancer (TNBC) cell lines (MDA-MB-231 and MDA-MB-468) and colon cancer cell line HCT-116 to investigate their structure−activity relationships. Most of these new complexes have shown appreciable activity, comparable to or significantly better than that of cisplatin in TNBC cell lines. R 4 substitution of the phenyl ring of the 2Hindazole ligand with methyl and isopropyl substituents showed increased potency in ruthenium(II) and iridium(III) complexes compared to that of their parent compounds in all cell lines. These novel transition metal-based complexes exhibited high specificity toward cancer cells by inducing alterations in the metabolism and proliferation of cancer cells. In general, iridium complexes are more active than the corresponding ruthenium complexes. The new Ir(III)-2H-indazole complex with an isopropyl substituent induced mitochondrial damage by generating large amounts of reactive oxygen species (ROS), which triggered mitochondrionmediated apoptosis in TNBC cell line MDA-MB-468. Moreover, this complex also induced G2/M phase cell cycle arrest and inhibited cellular migration of TNBC cells. Our findings reveal the key roles of the novel C−N-cyclometalated 2H-indazole-Ir(III) complex to specifically induce toxicity in cancer cell lines through contributing effects of ROS-induced mitochondrial disruption along with chromosomal and mitochondrial DNA target inhibition.