Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles

AbstractTo better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly

Synthesis and Evaluation of a Lead Binding Peptoid

Previous research had shown that peptides with the amino acid sequence GGGTNTLSNNGGG have an affinity for binding lead particles in solution. The aim of this project is to prepare a peptoid with binding affinity for lead. Initial results show a 27:1 lead to peptoid binding with an average 14% lead decrease in the presence of 1.31× 10-5 moles of the peptoid. The decrease in the lead concentration found in the solution coupled with the reusability of the peptoid resin are promising results for peptoid use in lead contaminated water supplies.https://digitalcommons.humboldt.edu/inrsep_posters/1004/thumbnail.jp

Synthesis and Evaluation of a Lead Binding Peptoid

https://digitalcommons.humboldt.edu/ideafest_posters/1113/thumbnail.jp

Analysis of Herbicides on Culturally Significant Plants Throughout Yurok Ancestral Territory

https://digitalcommons.humboldt.edu/inrsep_posters/1014/thumbnail.jp

Comparative Analysis of Anabaena Sensory Rhodopsin in Nanodiscs Containing Various Lipids.

Membrane proteins (MP) are crucial for cell pathways, but are difficult to study due to hydrophobicity. Nanodiscs (ND) provide a lipid bilayer mimetic enabling MP studies. To improve efficiency, we compared two ND assembly methods for detergent removal, and evaluated how lipid affects the MP, Anabaena Sensory Rhodopsin. ND were assembled with purified ASR, various lipids, Sodium Cholate and Apolipoprotein AI. Electrophoresis was utilized to evaluate complex formation. ASR was successfully isolated and assembled into ND. We found that the biobead method was faster and had less risk of loss, showing it to be the better method. ND provide a promising approach to therapeutics affecting MP

Nanodiscs Stabilize Anabaena Sensory Rhodopsin for Transcriptional Regulation Studies

Anabaena Sensory Rhodopsin (ASR) is a retinal containing membrane protein from Anabaena nostoc. ASR undergoes an orange-light induced conformational change from an all trans form to a 13-cis form, which is associated with the release of a bound transducer protein ASRT. It is proposed that the ASR/ASRT complex directly controls the transcription of phycocyanin (cpc-gene) and phycoerythrocyanin (pec-gene). In order to study this protein complex, ASR nanolipoprotein particles (nanodiscs) were assembled, which allows both ends of the ASR protein to be studied. These particles will enable the main goal of this research, which is to identify the mechanism of the ASR/ASRT control of transcription.https://digitalcommons.humboldt.edu/ideafest_posters/1008/thumbnail.jp

Activity of Novel Cellulases from Cow Rumen

Utilizing cellulase enzymes can enhance the production of biofuels. In this study, 14 cellulases identified through metagenomic analysis of cow rumen were expressed in E. Coli, purified using immobilized metal affinity chromatography (IMAC), and then assessed for enzymatic activity versus a control cellulase isolated from Aspergillus Niger. To do this, we evaluated the breakdown of carboxymethylcellulose (CMC) in a plate assay with Congo Red detection. 4 with high activity and 2 with low activity were selected for analyzing the effective pH on the enzymatic activity and expanding the research to kinetic analysis. Our results could inform new cellulase design and enhance biofuel production