Mingxuan Zhang

Email: mzhang@chemistry.umass.edu

Research:

I. Indirect Detection of Protein-Metal Binding: Interaction of Serum Transferrin with In3+ and Bi3+

Transferrins comprise a class of monomeric glycoproteins found in all vertebrates, whose function
is iron sequestration and transport. In addition to iron, serum transferrin also binds a variety of
other metals and is believed to provide a route for the in vivo delivery of such metals to cells. In
the present study, ESI MS is used to investigate interactions between human serum transferrin and
two nonferrous metals, indium (a commonly used imaging agent) and bismuth (a component of
many antiulcer drugs). While the UV-Vis absorption spectroscopy measurements clearly indicate
that both metals bind strongly to transferrin in solution, the metal-protein complex can be detected
by ESI MS only for indium, but not for bismuth. Despite the apparently low stability of the
transferrin-bismuth complex in the gas phase, presence of such complex in solution can be
established by ESI MS indirectly. This is done by monitoring the evolution of charge state
distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the
absence of the metal in solution. The anomalous instability of the transferrin-bismuth complex in
the gas phase is rationalized in terms of conformational differences between this form of transferrin
and the holo-forms of this protein produced by binding of metals with smaller ionic radii (e.g., Fe3+
and In3+). The large size of Bi3+ ion is likely to prevent formation of a closed conformation
(canonical structure of the holo-protein), resulting in a non-native metal coordination. It is
suggested that transferrin retains the open conformation (characteristic of the apo-form) upon
binding Bi3+, with only two ligands in the metal coordination sphere provided by the protein itself.
This suggestion is corroborated by the results of circular dichroism measurements in the near-UV
range. Since the cellular consumption of metals in the transferrin cycle critically depends upon
recognition of the holo-protein complex by the transferrin receptor, the noncanonical conformation
of the transferrin-bismuth complex may explain very inefficient delivery of bismuth to cells even
when a high dosage of bismuth-containing drugs is administered for prolonged periods of
time.

Relevent publication: J Am Soc Mass Spectrom 2004, 15, 1658–1664)

II. Understanding protein-metal binding interactions of transferrin
by ESI MS and hydrogen/deuterium exchange

Studies of backbone dynamics of transferrins (Tf) are vital for understanding the molecular processes
responsible for metal transport and sequestration. Hydrogen/deuterium exchange followed by mass
spectrometric detection (HDX MS) is an indispensable tool that provides valuable information on a variety
of dynamic events within protein without suffering from limitations typical of NMR spectroscopy, such as
those related to high molecular weight, presence of paramagnetic ligands (such as many metal ions), etc.
(1). Our initial studies of Tf dynamics by HDX MS relied on a combination of hydrogen exchange in
solution and protein ion fragmentation in the gas phase using a collision-induced activation, CAD.
However, straightforward application of this method to mapping structural flexibility of transferrin is
complicated due to the presence of multiple disulfide bonds within this protein (eight in the N-lobe alone).
Although facile cleavage of amide bonds is readily achieved in either FT ICR MS or Qq-
TOF MS, the amount of structurally diagnostic fragment ions is rather limited. The likely reason is the presence of multiple disulfides, which prevent physical separation of fragment ions
produced by cleavages of amide bonds in the gas phase.