Protein crystallization

Authors: Dr. Szymon Krzywda and Stanisław Wosicki, MSc

Aims

Purpose of this exercise

In this exercise you will search for optimal protein crystallization conditions. You will use a vapour diffusion method in hanging drops to crystallize chicken egg white lysozyme.

Practical information

For this practical, we shall meet next to the reception (close to the main entrance) of Collegium Chemicum
We will then walk together to the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznan, where the Center for Biocrystallographic Research (CBB) is located
The walk will take approximately 15 min. The address is: Noskowskiego 12/14
To locate the CBB on the map, go to this address and follow the "How to get to the Institute" link

Required reading

Read the following papers:

McPherson, A. Introduction to protein crystallization. Methods 34, 254–265 (2004).
Johnson, LN. The early history of lysozme. Nat Struct Mol Biol 5 (11): 942–944, (1998).
Voet, D; Voet, JG. Biochemistry. Fourth Edition: 517-525 (2011).

Lysozyme

Lysozyme catalyzes hydrolysis of 1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. This catalytic activity is nonspecifically targeted to the Gram positive bacterial cell wall and related with general nonspecific organism defense. Lysozyme is present in a number of secretion such as tears, saliva, human milk and mucus. Large amounts of lysozyme can be found in egg white.
In bacteriophages, lysozyme is used to break into and lyse the host bacterial cell.

Theory

Prepare the following theoretical issues:

protein crystallization,
methods and techniques of crystallization,
saturated and supersaturated solutions,
partial pressure,
phase diagrams,
entropy,
enthalpy.

Getting the most from the primary sequence

There are a few important biochemical properties of your protein you should know before setting up a crystallization experiment. Nowadays, the primary structure (sequence) is very often the only information you can go on. Despite its relative simplicity it can deliver a plethora of useful things.

The primary sequence of chicken egg white lysozyme is given below (LYSC_CHICK P00698 ):


      1          11         21         31         41         51         
      |          |          |          |          |          |          
    1                    KV FGRCELAAAM KRHGLDNYRG YSLGNWVCAA KFESNFNTQA    60
   61 TNRNTDGSTD YGILQINSRW WCNDGRTPGS RNLCNIPCSA LLSSDITASV NCAKKIVSDG   120
  121 NGMNAWVAWR NRCKGTDVQA WIRGCRL

You can calculate:

The number of tryptophan and tyrosine residues. The presence of these two amino acids endow protein with substantial absorbance at 280 nm. This allows to estimate protein concentration by spectrophotometry. You can calculate the lysozyme concentration using the equation:
A = ε · t · c / M

where: A – absorbance at λ = 280 nm; c - protein concentration [g · l^-1]; t – cell path length (1 cm); ε – molar extinction coefficient [l · mol^-1 · cm^-1]; M – protein molar mass [g · mol^-1].
The molar extinction coefficient can be predicted from the equation:
ε = n_W · 5690 + n_Y · 1280
where n_W, n_Y, are the number of tryptophan and tyrosine residues with their molar extinction coefficients, respectively. For more accurate value you should take into account the amount of phenylalanine and cystine (why not cysteine?) residues.
The number of cysteine residues. The odd number of cysteines would indicate that at least one cysteine is reduced and could be used for mercury derivative to get initial phases. You should consider the cellular location of the protein. As lysozyme is present in a number of secretion one can assume it's extracellular localization which is strongly supported by its 18 residues long signal peptide at the beginning of the sequence. (Note that the sequence starts with the residue number 19).
The number of methionine residues. Generally, more than one methionine per 100 amino acids will ensure the success of a selenomet MAD experiment.

Using the ExPasy server find ProtParam tool and calculate:

The molecular weight. Small value indicates it is a single domain protein. Multi-domain proteins (usually larger than 30 KDa) are more difficult to crystallize. Is lysozyme confirms this correlation?
The pI. When you have problems with solubility of your protein you should consider using a buffer which pH is farther away from the pI. Why?

If there is no computer connected to the Internet, do it at home.

Preparation

Preparation of lysozyme samples, crystallization plate and setting up crystallization experiment

Weigh out ~12 mg of liophilized lysozyme, dissolve in water to get 200 µL lysozyme solution at concentration of 40 mg/mL and 200 µL of 20 mg/mL.
Confirm the protein concentration spectrophotometrically. Do you know any other methods to estimate protein concentration? What are their advantages and drawbacks?
Put a thin layer of silicone grease at the edge of each well of the VDX plate, trying not to pollute inner walls.
Prepare a reservoirs' solutions in each well, according to the schedule given below. Use proper stock solutions and dilute to a volume of 1 mL.
On a siliconized glasses prepare crystallization drops by mixing 4µL of protein solution (concentration and pH are given in the first column of the table) and 4 µL of reservoir's solution (from the well). Remember, you have to prevent evaporation! After preparation of the crystallization drop close the well by putting the glass upside-down immediately (crystallization drop hangs inside the well). Carefully push down the glass, so that the grease sealed space of the well.
When the whole plate will be done, check under microscope how the crystallization drops looks and tightness. Especially pay attention to whether or not there was an immediate precipitation of proteins. Write down the observations. You can use the scoring sheet for example provided by Jena Bioscience.
Store the plate in the air-conditionning crystallization room (19°C) for a few days. After this time, check results of the experiment and update your scoring sheet. Write down observations and on their basis, determine the optimal conditions of crystallization of lysozyme and areas of concentration and pH, under which the precipitation of proteins occure.

	1	2	3	4	5	6
(A) Protein 1 c. protein 20 mg/mL, 50 mM acetate buf. pH 4.5	0.6 M NaCl	0.8 M NaCl	1.0 M NaCl	1.2 M NaCl	1.4 M NaCl	1.6 M NaCl
(B) Protein 2 c. protein 40 mg/mL, 50 mM acetate buf. pH 4.5	0.6 M NaCl	0.8 M NaCl	1.0 M NaCl	1.2 M NaCl	1.4 M NaCl	1.6 M NaCl
(C) Protein 3 c. protein 20 mg/mL, 50 mM acetate buf. pH 5.0	0.6 M NaCl	0.8 M NaCl	1.0 M NaCl	1.2 M NaCl	1.4 M NaCl	1.6 M NaCl
(D) Protein 4 c. protein 40 mg/mL, 50 mM acetate buf. pH 5.0	0.6 M NaCl	0.8 M NaCl	1.0 M NaCl	1.2 M NaCl	1.4 M NaCl	1.6 M NaCl

Crystals growth

Examination of the crystallization experiments

Examine the crystallization drops immediately after setup and at the beginning of the next practical. Make notes in the scoring sheet attached. You can use Johan Zeelen's numbering scheme to describe your results. The following pictures present some examples of crystallization drops you can obtain.

		clear drop
		non-protein particles e.g. pieces of glass, fibers from clothes
		mostly clear with some precipitant
		skin
		protein fully precipitated
		gelatinous precipitate
		phase separation
		spherulities
		microcrystals
		needles
		plates
		crystals

Images of your crystals

Report

Your Report

Your Report from this practical, as from all other practicals, should be completed and sent to me by email (szymon@amu.edu.pl) before the date of the following practical class.

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Last modification: Mar 14, 2014 (SW)