Protein Crystallization

Experiment 5: Protein Crystallization

2012-38353 | 2013-05949 | 2013-14905

Biochem 124.1 WCD – Group # 2 | Prof. Ciara Lim

October 12, 2016

1. Abstract

Biological macromolecules, such as proteins, undergo crystallization as a part of determining their tertiary structure. However, the chemical and thermodynamic properties specific to each protein, coupled with the physicochemical and biochemical environments were the protein is exposed, calls for a selection of reagents, pH, buffer, and precipitant concentrations in order for the protein to be classified well enough under X-ray crystallography. Using the hanging drop method, hen egg white lysozyme was crystallized using NaCl and polyethylene glycol (PEG) as precipitating agents under varying buffer and protein concentrations. Optimization resulted to ___mg/mL lysozyme in _____ resulted in the most satisfactory crystal growth.

1. Keywords: crystallization, lysozyme, hanging drop method, nucleation

1. Introduction

Crystallization is an important process in determining the three-dimensional structure of proteins and nucleic acids; this procedure is usually considered the bottleneck in structure determination. Several factors are involved in the crystallization process and can affect the probability of it occurring at all. These include the physical (e.g. temperature, pressure, time), chemical (e.g. pH, precipitant concentration, presence of cross-linkers), and biochemical (e.g. purity, chemical and genetic modifications) parameters (McPherson, 2004).

Moreover, proteins and nucleic acids are found to be more difficult to crystallize as compared to relatively smaller organic and inorganic due to a variety of reasons: 1) protein crystals have far fewer contacts in terms of molecular and are thus very weak, 2) protein purification is difficult despite the use of ultra-pure reagents, and 3) the diversity of chemical groups in proteins require highly specific chemical environments (Durbin & Feher, 1996). However, in terms of the methodology, three stages of growth are distinguished: nucleation, post-nucleation growth, and cessation of growth (Durbin & Feher, 1996).

Protein crystallization can be done in a variety of methods. The most widely-used involves vapor diffusion, where the protein solution is either a hanging or sitting drop that equilibriates against a reservoir containing precipitating agents found at either a higher or lower concentration than in the drop (Chayen, 2008). Popular precipitants include polyethylene glycol (PEG) and ammonium sulfate. Microbatch experiments are also usually employed in protein crystallization. Here, crystallization trials are dispensed and incubated in low-density paraffin oil, where they are protected from evaporation, airborne contamination, and shock, thus making the plates more easily transportable.

Whereas diffusion-based methods are dynamic systems in which conditions change throughout the crystallization process, in microbatch, the samples are mixed at their final concentration at the start of the experiment; thus, conditions are constant within a normal time frame of a crystallization experiment (1–3 weeks). There are usually no changes in drop volume or pH, and crystals do not usually dissolve (Chayen, 2008).

Hen egg white lysozyme (HEWL) is known to have a relatively easier crystallization process compared to other enzyme because 1) lysozymes, the major component of egg white, has a simpler composition, and 2) it is a rather small and stable globular protein whose enzymatic activity can be restored after dehydration and/or lyophilization (Lorber et.al., 1993).

In the experiment, a combination of the hanging drop and microbatch methods will be used to crystallize HEWL under different chemical conditions. It will also help determine the optimum buffer concentration, pH, and precipitant concentration for maximum crystallization.

1. Methodology

The following protein (hen egg white lysozyme) stock solutions were prepared and crystallized using two set-ups:

Sample 1: 30 mg/mL in 50 mM NaOAc, pH 4.5

Sample 2: 60 mg/mL in 50 mM NaOAc, pH 4.5

Sample 3: 75 mg/mL in 50 mM NaOAc, pH 4.5

For Set-Up A, samples 2 and 3 were used with the following crystal/well solutions (1 mL/well):

Solution A: 13% w/v NaCl in 0.1M NaAC, pH 4.5

Solution B: 6.25% w/v NaCl in 0.1M NaAC, pH 4.5

Solution C: 3.125% w/v NaCl in 0.1M NaAC, pH 4.5

Solution D: 1.625% w/v NaCl in 0.1M NaAC, pH 4.5

Using the hanging drop method, 5 µL of protein solution was mixed with 5 µL of the reservoir solution and placed in the crystal wells as shown below:

For Set-Up B, samples 1 and 2 were used with the following crystal/well solutions (1 mL/well):