Topic
RiAFP
About: RiAFP is a research topic. Over the lifetime, 5 publications have been published within this topic receiving 186 citations.
Papers
TL;DR: A simple and effective procedure to isolate antifreeze proteins (AFPs) from the hemolymph of larvae of the longhorn beetle Rhagium inquisitor is described, and some characteristics of their structures are presented.
Abstract: We describe a simple and effective procedure to isolate antifreeze proteins (AFPs) from the hemolymph of larvae of the longhorn beetle Rhagium inquisitor , and present some characteristics of their structures. Several AFPs were isolated from the hemolymph of this species by heat and acid extraction followed by cation exchange. The hemolymph contains at least six AFPs ranging in size from 12.5 to 12.8 kDa. Of these, three were separated to purity by the ion exchange step, as indicated by mass spectrometry. The remaining three forms were further separated by size exclusion chromatography, but could not be isolated to purity. All AFPs in the hemolymph of this species appears to have isoelectric points above 8.00. The dominant form, RiAFP H4 , was purified by the ion exchange step. Its amino acid composition reveals a lower level of cysteine and a higher level of threonine, arginine, alanine and glycine than seen in other insect AFPs. Its trypsin fingerprint does not match that of any known protein. It interacts with ice both in the anionic and cationic state.
36 citations
TL;DR: The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs.
Abstract: Antifreeze proteins (AFPs) are a specialized evolutionary adaptation of a variety of bacteria, fish, arthropods and other organisms to inhibit ice-crystal growth for survival in harsh subzero environments. The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs. For this purpose, RiAFP was overexpressed in Escherichia coli, purified and crystallized at 293 K using a combination of 23% PEG 3350 and 0.2 M ammonium sulfate as a precipitant. X-ray diffraction data were collected to 1.3 A resolution using a synchrotron-radiation source. The crystals belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 46.46, c = 193.21 A.
12 citations
TL;DR: A genetic algorithm for the in silico design of AFP mutants with improved antifreeze activity, measured as the predicted thermal hysteresis at a fixed concentration, ΔTC is presented.
Abstract: Antifreeze proteins (AFPs) are of much interest for their ability to inhibit ice growth at low concentrations. In this work, we present a genetic algorithm for the in silico design of AFP mutants with improved antifreeze activity, measured as the predicted thermal hysteresis at a fixed concentration, ΔTC. Central to the algorithm is our recently developed neural network method for predicting ΔTC from molecular simulations [Kozuch et al., PNAS, 115, 13252 (2018)]. Applying the algorithm to three structurally diverse AFPs, wfAFP, rQAE, and RiAFP, we find that significantly improved mutants are discovered for rQAE and RiAFP. Testing of the optimized mutants shows an increase in ΔTC of 0.572 ± 0.11 K (262 ± 50.6%) and 1.33 ± 0.14 K (39.9 ± 4.19%) over the native structures for rQAE and RiAFP, respectively. Structural analysis of the optimized mutants reveals that the algorithm is able to exploit two pathways for enhancing the predicted antifreeze activity of the mutants: (1) increasing the local order of surface waters by encouraging the formation of internal water channels in the protein and (2) increasing the total ice-binding area by improving the planar structure of the ice-binding surface. Additionally, analysis of all mutants explored by the algorithm reveals that a subset of residues, mainly nonpolar, are particularly helpful in improving antifreeze activity at the ice-binding surface.
9 citations
TL;DR: This work reports on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle that has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects.
TL;DR: The crystal structure of a potent beetle antifreeze protein, the 13-kDa RiAFP, is the largest determined ab initio without heavy atoms, and its ordered waters suggest a molecular basis for ice binding.
Related Topics (5)
Performance Metrics
| Year | Papers |
|---|---|
| 2020 | 1 |
| 2013 | 1 |
| 2012 | 1 |
| 2011 | 1 |
| 2005 | 1 |