Rigidity Analysis of Protein Molecules

Intrinsic flexibility of protein molecules enables them to change their 3D structure and perform their specific task. Therefore, identifying rigid regions and consequently flexible regions of proteins has a significant role in studying protein molecules' function. In this study, we developed a kinematic model of protein molecules considering all covalent and hydrogen bonds in protein structure. Then, we used this model and developed two independent rigidity analysis methods to calculate degrees of freedom (DOF) and identify flexible and rigid regions of the proteins. The first method searches for closed loops inside the protein structure and uses Gr€ ubler-Kutzbach (GK) criterion. The second method is based on a modified 3D pebble game. Both methods are implemented in a MATLAB program and the step by step algorithms for both are discussed. We applied both methods on simple 3D structures to verify the methods. Also, we applied them on several protein molecules. The results show that both methods are calculating the same DOF and rigid and flexible regions. The main difference between two methods is the run time. It's shown that the first method (GK approach) is slower than the second method. The second method takes 0.29 s per amino acid versus 0.83 s for the first method to perform this rigidity analysis

Stabilisation and encapsulation studies on xylanase for animal feed improvement

Pig and poultry feeds contain materials that are derived from plant and animals. Most of the plant materials are indigestible because they contain non-starch polysaccharides and as a result the animal suffers from anti-nutritional effects. To reduce the anti-nutritional effects, a number of enzymes, including xylanase, are added to the feed to break down the non-starch polysaccharides. Prior to ingestion, the feed must be processed to destroy any microbial contaminants. As a consequence of this action, the enzymes become inactivated due to the high temperatures of processing. The aim of this project was to improve the quality of the feed by preventing the degradation of enzymes during processing. In order to carry out a thorough investigation to improve the stability of xylanase, a full characterisation profile was determined first. The denaturation of xylanase when exposed to stressful conditions was monitored by circular dichroism spectroscopy. In most occasions, in the presence of low molecular weight additives, xylanase had enhanced activity and improved structural stability. Stabilisation by immobilisation on two support materials such as modified silica and chitosan improved the thermal stability of xylanase. Conditions typically reproduced within a processing cycle were used to investigate the stability of the immobilised xylanase. Microencapsulation of xylanase was also carried out by spray drying with stabilising polymers and by phase separation methods. The enzyme activities following each formulation were determined. The morphology of the microspheres produced was examined using scanning electron microscopy

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform