Iglesias, Eloisa Marie C.

Cell & Molecular Biology

BS Biology 4-2 Critique Paper 1

De Novo Proteins with Life-Sustaining Functions Are Structurally Dynamic

Grant S. Murphy, Jack B. Greisman and Michael H. Hecht

From the previous studies of designing proteins, it was assumed that a well-ordered
structure was an essential prerequisite for protein function. Because of this assumption, protein
designers began to consider the possibility of devising novel proteins that bind targets and/or
catalyze reactions. In recent years, many studies have shown that many natural proteins
responsible for essential cellular functions are intrinsically disordered and/or dynamic. This
study, on the other hand, questioned these assumptions by probing the structural and
biophysical properties of several -helical proteins. To achieve this, the researchers did several
experimental techniques. Protein structure prediction simulations were performed using the
Rosetta macromolecular modeling software fragment assembly protocol. Proteins were
expressed in E. coli BL21 (DE3) pLysS cells. Proteins were purified using immobilized metal
affinity chromatography (IMAC). Also, CD data were collected on a Chirascan CD spectrometer
(Applied Photophysics). NMR spectra were collected on an 800-MHz AVANCE III HD
spectrometer (Bruker) with a 5-mm cryoprobe. A Superdex 75 5/150 column (GE Healthcare)
was used for analytical SEC (size-exclusion chromatography). There were seven SynRescue
proteins that were investigated. Their properties were compared to three control proteins from
the second-generation library. Computational structure prediction simulations were performed
for each of the SynRescue proteins and the control proteins. It is found out that the SynRescue
proteins are -helical and thermostable and that they denature reversibly. However, 1H15N
HSQC NMR experiments demonstrate that their structures are dynamic and undergo kinetic
exchange on an intermediate timescale. SEC indicates that the SynRescue proteins do not form
long-lived monomeric structures but instead form extended dimers that are kinetically unstable
on the timescale of the chromatography experiments. In addition, they have also identified three
features that may have favored the formation of extended -helical hairpin that assemble into
domain-swapped dimers: 1) breaking the hydrophobic register 2) preventing favorable buried
polar interactions and 3) interrupting helix propensity. They also found out that a well-ordered
structure is not a pre-requisite for biological function.

The article is quite long and its contents are somehow difficult to understand, especially
if the reader is not well-equipped with knowledge regarding the topic, which is a complex one. It
is very much filled with too many information and it may have caused confusion in my head.
However, I believe that the article is a very good one for in fact, it was published in Elsevier
Journal of Molecular Biology last 2015. As a recent publication, it has a significant contribution in
the field of science. It has provided a new factual information particularly to the field of molecular
biology. But unlike any other journal articles, this one lacked the conclusion part and the
methodology was placed in the last part, before the references but even if this is the case, the
authors described the methods comprehensively and adequately. The results of the experiment
were presented properly, the data were organized and there were graphs and diagrams. The
results were interpreted correctly and were supported by other researches and literature. As a
whole, this article was written very well and it can be used as a reference or a guide for future
researches.