fd Coat Protein Structure in Membrane Environments:
Structural Dynamics of the Loop Between the
Hydrophobic Trans-Membrane Helix and the
Amphipathic In-Plane Helix
F.C.L.AlmeidaandS.J.Opella*
Department of Chemistry
University of Pennsylvania
Philadelphia
PA 19104-6323, USA
By performing multidimensional solution NMR experiments on micelle
samples it was possible to determine the structure of the membrane-
bound form of fd coat protein based on short-range distance and dihe-
dral angle constraints using distance geometry and simulated annealing
calculations. Its dynamics were described by
15
N relaxation measure-
ments (T
1
, T
2
, heteronuclear nuclear Overhauser enhancement (NOE))
®tted with the Lipari±Szabo model-free formalism adapted for the trans-
membrane and in-plane helices of a membrane protein. The overall corre-
lation time of the protein in micelles was found to be $9 ns, and the
local motion of each backbone N-H vector was described by an order
parameter and an effective correlation time. The 50 residue protein has
an amphipathic a-helix (residues 7 to 16) and a hydrophobic a-helix (resi-
dues 27 to 44), which were found to be approximately perpendicular on
the basis of NOEs in the residues that connect the two helices. The resi-
dues connecting the helices are of particular interest in membrane pro-
teins, and in this case the loop consists of two turns. The relaxation data
show the presence of an extra motion in the amphipathic a-helix on the
nanosecond timescale and additional ¯exibility of several residues in the
loop connecting the two helices.
# 1997 Academic Press Limited
Keywords: membrane protein; NMR spectroscopy; molecular dynamics
simulations; fd coat protein; protein dynamics
*Corresponding author
Introduction
fd coat protein is a typical membrane protein as
judged by its biological, chemical, and structural
properties. It is strongly associated with the host
membrane of infected cells, where it is stored prior
to the assembly of ®lamentous bacteriophage par-
ticles which are extruded into the periplasm with-
outcelllysis(Russel,1991).Itisveryhydrophobic
and completely insoluble in water when separated
from virus particles or membranes. In membrane
environments the protein has two distinct a-helical
segments, a short nine residue amphipathic helix
that lies in the plane of the bilayer and a long 18
residuetrans-membranehydrophobichelix(Shon
etal.,1991a;McDonnelletal.,1993).Thestructure
and dynamics of the nine residues located between
the two helices are of interest and are the principal
subjects of this paper. The N and C-terminal re-
gions of the protein in membrane environments
are mobile, although there is some evidence that
helical secondary structure persists through to the
Cterminus(McDonnelletal.,1993).Thecoatpro-
tein in virus particles is a single, somewhat dis-
torted a-helix with only the ®rst four to ®ve
residuesmobileandunstructured(Opellaetal.,
1987;Glucksmanetal.,1992;Marvinetal.,1994).
The most striking difference between the structures
of the coat protein in virus particles and membrane
environments is the angle between the amphi-
pathic and hydrophobic helices, which is very
small when the protein is in virus particles and
approximately 90
when it is in membranes. This
means that the residues connecting the two helices
Abbreviations used: MD, molecular dynamics; NOE,
nuclear Overhauser enhancement; RMS, root-mean-
squared; RMSD, root-mean-squared-deviation; NMR,
nuclear magnetic resonance; HMQC, heteronuclear
multiple quantum correlation; NOESY, NOE
spectroscopy; TOCSY, total correlated spectroscopy;
ppm, parts per million.
J. Mol. Biol. (1997) 270, 481±495
0022±2836/97/280481±15 $25.00/0/mb971114 # 1997 Academic Press Limited