The Aggregate State Of The Protein Crystal Growth

It is the aim of the crystal growth researchers to find the optimized condition ofprotein crystal growth. Because different proteins have their own growth conditions, itis necessary to give a criterion of protein crystal growth. There are little studies ofthese criterions, and most of the studies have mainly focused on the post process ofnucleation process. The change of protein aggregates state (including the size, themorphology and the structure) in the fore process of nucleation of protein solution candirectly influence the nucleation process. The aggregates appeared before proteinnucleation process is mass-fractal, while they are ordered after nucleation process. So,it is important to study the aggregates before and after nucleation process anddetermine the crystal growth condition by the state of aggregates.In this dissertation aggregates in protein solution have been studied by Dynamiclight scattering and Atom Force Microscope. Concanavalin A has been chosen to be amodel protein. The change of state of aggregates in Con A solution during both thenucleation process and protein crystal growth has been studied under bothcrystallizable condition and noncrystallizable condition. Moreover, the relationbetween the change of the state of aggregates and the con A concentration,temperature of protein solution have been studied.We have studied the change of aggregates of concanavalin A in solution by DLS1 hour after the crystal growth starting. The concentrations of con A used in thetemperature are from 0.06mg/ml to 1.6mg/ml. Different temperature, 15℃, 20℃,25℃, have been used. Every sample has been repeatedly measured at least ten times.The result has been analyzed by NNLS and then the mean diameter of aggregates hasbeen obtained taking the average. The precipitation solution was 0.7mg/ml NaClsolution. After DLS experiments, the concanavalin A solution with certainconcentration was transformed to silicon wave every day during the crystal growthprocess and were observed by AFM under crystallizable condition andnoncrystallizable condition. The crystallizable condition and noncrystallizablecondition are: con A concentration 1.1mg/ml and 0.2mg/ml, respectively. The proteinsolution and precipitation solution were mixed the same volume. During all the AFMexperiments the temperature is 20℃, the humidity is 50-60%, and the adsorption timeof con A solution onto the silicon wave is 7 minutes.The followings are our experiment results: in the con A crystal growth process,at 15 degree, the size of the aggregates of con A in solution decreased starting from120nm as the con A concentration increased. However, after the concentrationincreased to 0.79mg/ml, the size of the aggregates constant almostly around 74nm. At20 degree, the size of the aggregates of con A in solution decreased starting from111nm as the con A concentration increased. After the concentration increased to0.45mg/ml, the size of the aggregates is constant almostly around 74nm. At 25 degree,the size of the aggregates of con A in solution decreased starting from 106nm as thecon A concentration increased. After the concentration increased to 0.84mg/ml, thesize of the aggregates is constant almostly around 74nm. In the non-growth solution,the change of the aggregates size of the concanavalin A in solution is disorderly as thecon A concentration increased. In the con A crystal growth process, the percentage ofthe aggregates (size from10-20nm) decreased under the crystallizable condition, whileit increased under noncrystallizable condition.According to our experiments, the following conclusions can be drawn: in thenucleation process, in the growth solution the size of the aggregates of theconcanavalin A in solution decreased as the con A concentration increased. However,after the size of aggregates decreased to around 70nm, it kept constant almostly.Around 70nm is the size of elementary units of con A. So most of aggregates in thesolution is elementary units. In the non-growth solution, the change of the aggregatesstate of the concanavalin A in solution is disorderly as the con A concentrationincreased. The change of the temperature doesn't influence essentially the aggregatesstate in the solution, and the charge tendency has not been changed. Temperature onlyinfluenced the minimum con A concentration at which the aggregates started to keepconstant. In the con A crystal growth process, the percentage of the elementary unitsdecreased under the crystallizable condition, while it increased undernoncrystallizable condition. The aggregates state of protein solution is important tothe protein crystal growth, especially in 1 hour after the crystal growth starting.