Topics Covered
Background
Denaturation of Proteins
Dynamic Light Scattering for Characterisation
of Proteins
The Protein Melting Point
Factors Affecting the Melting Point of Proteins
The Malvern Zetasizer Nano System
Background
Proteins are composed of polypeptide
chains, synthesized within the cell from a pool of 20 different amino
acid types. In contrast to manmade and random coil biological polymers,
the protein's polypeptide chains are folded into unique 3-dimensional
structures in the natured state. These structures are stabilized by
a combination of electrostatic and hydrophobic interactions, along
with the formation of multiple hydrogen bonds between the side chains
of the amino acids within the structure.
Denaturation
of Proteins
Proteins were evolved to perform specific functions within
biological systems, such as catalysing reactions and transporting small molecules
or ions. This functionality necessitates a large degree of flexibility within
the structure of the protein. As such, the interactions stabilizing the protein
structure are just sufficient to maintain the structure within a narrow range
of environmental conditions. If solution conditions are outside of this range,
e.g. through a change in the pH or temperature, entropically driven denaturation
or unfolding can quickly occur. When denaturation occurs, the small size of
the protein is increased to a value consistent with a random coil polymer of
the same molecular weight. In the absence of chaotropic (aggregation prohibiting)
agents, inter-polymer hydrophobic interactions can quickly lead to non-specific
aggregation of the denatured polypeptide chains. A representation of the denaturation
process, along with the subsequent change in size is shown in Figure 1.
Figure 1. Schematic detailing the protein denaturation
process and the subsequent change in hydrodynamic size indicated by the diameter
of the solid circle.
Dynamic Light
Scattering for Characterisation of Proteins
Dynamic light scattering (DLS) has
long been used as a protein characterization tool. In DLS, the Brownian
motion of the particles is measured, and the hydrodynamic size is
calculated using the Stokes-Einstein equation. The sensitivity of
DLS is sufficient to distinguish different oligomeric and quaternary
protein states, and is ideally suited for monitoring the stability
of the protein structure to denaturing conditions.
The Protein Melting
Point
The protein melting point (TM) is defined as the temperature
at which the protein denatures. The change in size that accompanies the protein
denaturation is easily identified using DLS techniques. Consider Figure 2 for
example, which shows the temperature dependent Z-Average diameter and scattering
intensity for Ovalbumin in phosphate buffered saline (PBS) measured with a Zetasizer Nano ZS. At temperatures less than 71°C, the size
and scattering intensity are constant, suggesting a stable tertiary structure.
At 71°C and higher, both the size and scattering intensity increase exponentially
with temperature, indicating the presence of denatured aggregates.
Figure 2. Melting point trace and hysteresis for Ovalbumin
in PBS.
Factors Affecting
the Melting Point of Proteins
Because of the unique primary sequence of amino acids associated with specific
protein types, melting points can differ significantly. Solutions conditions,
e.g. pH and salt concentration, and post translational modifications, e.g. glycosylation,
can also have a marked influence on the stability of the protein structure and
hence the melting temperature. Melting point measurements then are an important
step in the total characterization of the target protein, particularly if the
protein is destined for integration into a pharmaceutical product or formulation.
Figure 3 shows the melting point traces for a series of proteins in PBS. The
automated measurements were collected with a Zetasizer Nano ZS, using a 1°C incremental temperature ramp
and a 3 minute equilibrium time at each measurement temperature.
Figure 3. Melting point traces and TM values for a series
of proteins in PBS.
The Malvern
Zetasizer Nano System
The Zetasizer Nano ZS from Malvern
Instruments is the first commercial instrument to include the
hardware and software for combined dynamic, static, and electrophoretic
light scattering measurements, giving the researcher a wide range
of sample properties, including the size, molecular weight, and zeta
potential. The system was specifically designed to meet the low concentration
and sample volume requirements typically associated with pharmaceutical
and biomolecular applications, along with the high concentration requirements
for colloidal applications. Satisfying this unique mix of requirements
was accomplished via the integration of a backscatter optical system
and the design of a novel cell chamber. As a consequence of these
features, the Zetasizer Nano ZS specifications for sample size and concentration
exceed those for any other commercially available DLS instrument,
with a size range of 0.6nm to 6um, and a concentration range of 0.1mg/mL
lysozyme to 40% w/v. As an added bonus, the Zetasizer Nano ZS hardware is self optimizing, and the software
includes a unique "one click" measure, analyze, and report feature
designed to minimize the new user learning curve.