Increased Protein Density Research Articles

Curvature sensing of curvature-inducing proteins with internal structure.

Many types of peripheral and transmembrane proteins can sense and generate membrane curvature. Laterally isotropic proteins and crescent proteins with twofold rotational symmetry, such as Bin/Amphiphysin/Rvs superfamily proteins, have been studied theoretically. However, proteins often have an asymmetric structure or a higher rotational symmetry. We studied theoretically the curvature sensing of proteins with asymmetric structures and structural deformations. First, we examined proteins consisting of two rodlike segments. When proteins have mirror symmetry, their sensing ability is similar to that of single-rod proteins; hence, with increasing protein density on a cylindrical membrane tube, a second- or first-order transition occurs at a middle or small tube radius, respectively. As asymmetry is introduced, this transition becomes a continuous change and metastable states appear at high protein densities. Protein with threefold, fivefold, or higher rotational symmetry has laterally isotropic bending energy. However, when a structural deformation is allowed, the protein can have a preferred orientation and stronger curvature sensing.

Binding and wrapping of E. coli single stranded binding protein (SSB) C-terminal tail variants measured using force spectroscopy.

E. coli single-stranded DNA (ssDNA) binding protein (SSB) binds and wraps ssDNA in multiple conformations, plays an important role in DNA replication and repair, and has been utilized in biochemical applications such as increasing PCR specificity and yield. Structurally, SSB consists of an oligonucleotide binding domain (OB) that forms stable tetramers, and an unstructured C-terminal tail with an acidic tip. We use optical tweezers to measure the structure of a long ssDNA saturated with SSB. While low SSB concentrations stably contract the ssDNA with large amplitude, higher concentrations force bound SSB into a less wrapped conformation, decreasing binding site size and extending the substrate. This is consistent with the conformation of the SSB-ssDNA complex regulated through competitive binding, wherein increased protein density destabilizes higher order wrapped states and promotes the dissociation of excess SSB from the substrate. To determine the role of the C-terminal domain in this regulatory behavior, we measure the binding and wrapping of two C-terminal tail truncations, lacking either the 8-residue acidic tip (SSB-∆C8) or the entire C-terminal tail (SSB-OB). We find that removal of the acidic tip increases the binding affinity, which further increases with the removal of the whole C-terminal tail. Removal of the entire C-terminal tail results in increased biphasic binding as well as a greatly reduced direct dissociation rate. These results suggest that the C-terminal tails do not facilitate the SSB-SSB interactions that are responsible for cooperativity, but instead moderate the binding to and wrapping of ssDNA by competing for OB binding sites.

Emerging enzyme surface display systems for waste resource recovery.

The current century marks an inflection point for human progress, as the developed world increasingly comes to recognize that the ecological and socioeconomic impacts of resource extraction must be balanced with more sustainable modes of growth that are less reliant on non-renewable sources of energy and materials. This has opened a window of opportunity for cross-sector development of biotechnologies that harness the metabolic problem-solving power of microbial communities. In this context, recovery has emerged as an organizing principal to create value from industrial and municipal waste streams, and the search is on for new enzymes and platforms that can be used for waste resource recovery at scale. Enzyme surface display on cells or functionalized materials has emerged as a promising platform for waste valorization. Typically, surface display involves the use of substrate binding or catalytic domains of interest translationally fused with extracellular membrane proteins in a microbial chassis. Novel display systems with improved performance features include S-layer display with increased protein density, spore display with increased resistance to harsh conditions, and intracellular inclusions including DNA-free cells or nanoparticles with improved social licence for in situ applications. Combining these display systems with advances in bioprinting, electrospinning and high-throughput functional screening have potential to transform outmoded extractive paradigms into 'trans-metabolic" processes for remediation and waste resource recovery within an emerging circular bioeconomy.

Increased Protein Density During Weight Loss Is Correlated with Reduced Abdominal Obesity and Body Mass Index and Improved Body Composition

Abstract Objectives There is a clear link between abdominal obesity and chronic diseases. Dietary changes leading to substantial weight loss reduce obesity and improve health; however, no viable dietary treatment program exists that produces clinically significant, cost-effective, and sustainable weight loss. To test the hypothesis that a diet dense in lean proteins and fiber is inversely associated with abdominal obesity while maintaining skeletal muscle mass (SMM), we evaluated the correlation between mean protein and fiber density and changes in BMI, waist circumference and SMM during weight loss. Methods Thirty adult males and females participated in this ongoing, 2-year dietary weight loss program. The Individualized Dietary Improvement Program focused on reducing caloric intake and increasing protein (7–11 g/100 kcal) and fiber (1.8–3.2 g/100 kcal) density to desired ranges. Participants attended 19 group educational sessions, 3 individual counseling appointments, self-weighed daily, and submitted monthly 24-hour dietary recalls. BMI, waist circumference and body composition (InBody) measurements were collected at baseline and after 6 months. Results At 6 months, 25 participants (24–70y) remained in the study with 18 completing all body measurements. Mean weight loss (n = 25) was −2.2 ± 0.5 BMI points (–5.2 ± 1.3% of initial body weight) and mean waist circumference reduction (n = 18) was −6.5 ± 1.3 cm from baseline. Significant increases in protein and fiber density were seen from baseline to month 2 (P < 0.05). There were direct inverse associations between mean protein density and both reduced waist circumference (P < 0.01) and reduced BMI (P < 0.01). Fiber intake had no significant impact on weight loss, and maintenance of SMM did not significantly correlate with mean protein density. However, only 11.0 ± 3.2% of weight lost was due to the loss of SMM, supporting the efficacy of the program. A significant positive correlation (P < 0.01) existed between fat mass loss and protein density, with 74.3 ± 4.7% of excess body fat accounting for total weight lost. Conclusions Increased protein density correlates with accelerated loss of fat mass, greater reductions in abdominal adiposity, and may protect SMM from degradation during weight loss. Funding Sources This research is supported by the NIBIB of the NIH and the USDA NIFA.

E. coli Single Stranded Binding Protein (SSB) Self-Regulates Wrapping of SSDNA through Competitive Binding

E. coli single-stranded DNA (ssDNA) binding protein (SSB) is a stable homotetramer that wraps ssDNA in multiple conformations depending on protein concentration and solution conditions. SSB plays an important role in DNA replication and repair and has been utilized in biochemical applications such as increasing PCR specificity and yield. Here, we use an optical tweezers system to measure the binding and wrapping of a single ssDNA substrate by SSB. At saturating concentrations (>1 nM), SSB binds to ssDNA in a biphasic manner, in which a rapid contraction of the ssDNA is followed by a slower partial elongation. Subsequent removal of free SSB from solution results in further contraction of the ssDNA and addition of free SSB again elongates the ssDNA. Oscillations between the two states are controlled by free protein concentration and are repeatable over many cycles. In contrast, low SSB concentrations (<1 nM) stably contract the ssDNA with increased amplitude. We show that these results are consistent with a generalized model in which the conformation of the SSB-ssDNA complex is regulated through competitive binding, where increased protein density destabilizes higher order wrapped states and promotes the dissociation of excess SSB from the substrate. Furthermore, the ability of SSB to form stable tetramers and interactions between the disordered C-terminal tails are critical to these functions. Our results are consistent with a functional model of SSB that maximizes its ability to protect transiently exposed ssDNA without inhibiting replication and repair processes.

Molecular AFM imaging of Hsp70-1A association with dipalmitoyl phosphatidylserine reveals membrane blebbing in the presence of cholesterol.

Hsp70-1A-the major stress-inducible member of the HSP70 chaperone family-is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5μg/ml (7nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature.

Restoring the biophysical properties of decellularized patches through recellularization.

Various extracellular matrix (ECM) scaffolds, isolated through decellularization, were suggested as ideal biomimetic materials for 'Functional tissue engineering' (FTE). The decellularization process comprises a compromise between damaging and preserving the ultrastructure and composition of ECM-previously shown to affect cell survival, proliferation, migration, organization, differentiation and maturation. Inversely, the effects of cells on the ECM constructs' biophysical properties, under physiological-like conditions, remain still largely unknown. We hypothesized that by re-cellularizing porcine cardiac ECM (pcECM, as a model scaffold) some of the original biophysical properties of the myocardial tissue can be restored, which are related to the scaffold's surface and the bulk modifications consequent to cellularization. We performed a systematic biophysical assessment of pcECM scaffolds seeded with human mesenchymal stem cells (MSCs), a common multipotent cell source in cardiac regenerative medicine. We report a new type of FTE study in which cell interactions with a composite-scaffold were evaluated from the perspective of their contribution to the biophysical properties of the construct surface (FTIR, WETSEM™) and bulk (DSC, TGA, and mechanical testing). The results obtained were compared with acellular pcECM and native ventricular tissue serving as negative and positive controls, respectively. MSC recellularization resulted in an inter-fiber plasticization effect, increased protein density, masking of acylated glycosaminoglycans (GAGs) and active pcECM remodelling which further stabilized the reseeded construct and increased its denaturation resistance. The systematic approach presented herein, therefore, identifies cells as "biological plasticizers" and yields important methodologies, understanding, and data serving both as a reference as well as possible 'design criteria' for future studies in FTE.

A novel approach to characterize the binding orientation of lysozyme on ion-exchange resins

Much work has been done to qualify and quantify chromatographic adsorption and transportation mechanisms in different adsorber materials. An important aspect in all studies is the understanding of the binding mechanism between protein and resin on a molecular level in order to optimize processes on the level of adsorber design. We established a method to determine the binding orientation of lysozyme for different materials under various experimental conditions enabling us to observe changes in the mode of adsorption. We varied the protein load of two different adsorber types, Source 15 S, a conventional cation exchange resin and EMD Fractogel SO 3, a tentacle-type cation exchanger. We found similar preferential binding sites for the interaction between lysozyme and the surface of these adsorbers at low surface coverage, however, the tentacle adsorber exhibited multi-point binding whereas the binding on Source was limited to one binding site only. With increasing protein density on the surface, lysozyme rotates from a space-consuming side-on to a space-saving end-on orientation on Fractogel, explaining a higher maximum binding capacity for Fractogel. This re-orientation could not be observed for Source.

The effect of protein density of food on food intake and nutritional status of tumor‐bearing rats

Current clinical practice emphasizes increasing calorie and protein intake to abate the nutritional decline that frequently occurs in cancer patients. Using an animal model of tumor-induced anorexia, we found that increasing the protein density of food resulted in a net increase in protein intake, but a decrease in the food intake of both healthy and tumor-bearing animals. The increased protein intake did not affect the nutritional status of tumor-bearing animals as indicated by body weight or serum levels of total protein, insulin, or insulin-like growth factor 1. These data suggest that factors regulating feeding responses to increased protein density of food are intact in hypophagic tumor-bearing rats, and that increased protein intake does not influence plasma levels of hormones requisite for protein synthesis. These data may partially explain why interventions to improve the nutritional intake of cancer patients have marginal effects on body weight, accrual of lean body mass, or synthesis of visceral proteins. © 1997 John Wiley & Sons, Inc. Res Nurs Health 20: 131–138, 1997

Formulation of Broiler Finishing Rations by Quadratic Programming

AbstractLeast cost feed mix by linear programming (LP) is a standard economic analysis in the poultry industry. A significant body of nutrition knowledge is now contained in the constraint set of industry LP models. This knowledge might be merged into an improved economic model that contains production response information. Analysis using a quadratic programming model indicated that a leading broiler firm could have improved economic efficiency by increasing protein density and reducing energy density of broiler finisher feed. If applicable industry wide, similar savings could be as high as $120 million per year.