Transcript Document

General Principles of Membrane
Protein Folding and Stability
Membrane Protein Structural Motifs: Basic Principles. One broad thermodynamic
principle underlies the structure and stability of membrane proteins: The
thermodynamic cost of transferring charged or highly polar uncharged compounds into
the oil-like hydrocarbon interior of bilayer membranes is very high. This has two
consequences. First, most of the amino acid sidechains of transmembrane segments
must be non-polar (e.g. Ala, Val, Leu, Ile, Phe). Second, the very polar CONH groups
(peptide bonds) of the polypeptide backbone of transmembrane segments must
participate in hydrogen bonds (H-bonds) in order to lower the cost of transferring them
into the hydrocarbon interior.
This H-bonding is most easily
accomplished with alpha-helices
for which all peptide bonds are
H-bonded internally. It can also
be accomplished with beta-sheets
provided that the beta-strands
form closed structures such as the
beta-barrel. All membrane proteins
of known three-dimensional structure
adhere to these principles.
Examples of the two known
structural motifs, bacteriorhodopsin
and a porin, are shown below.
How Membrane Proteins Are Assembled. Constitutive membrane
proteins, i.e. those that are encoded in a normal cell's genome and are
responsible for vital physiological activities, are assembled by means of a
complex process involving synthesis of membrane proteins by ribosomes
attached transiently to a complex of proteins referred to as a translocon
located within the cell membrane (below). This translocon provides a
transmembrane "tunnel" into which the newly synthesized protein can be
injected. After the synthesis is complete, the ribosome disengages from the
translocon (which enters a closed state) and the protein is released into the
membrane bilayer where it assumes (in an unknown way) its final folded
three-dimensional structure.
Non-constitutive membrane proteins are generally proteins such as toxins and
antimicrobial peptides that incorporate themselves into membranes of cells without
passing through the constitutive pathway. A good example is staphylococcal alphahemolysin (below) whose crystallographic structure was solved by Song et al. Nonconstitutive membrane proteins usually insert themselves into target membranes by
physicochemical processes and are therefore of particular value in understanding
membrane protein stability.
1. The Unfolded (Virtual) Reference State. The reference state is taken as the
unfolded protein in the interface. However, as far as we know, one cannot actually
achieve this state with constitutive membrane proteins because of the solubility
problems nor with small non-constitutive membrane-active peptides because binding
usually induces secondary structure (partitioning-folding coupling). Thus, as is often
the case in solution thermodynamics, the reference state must be a virtual one. This is
defined it by means of an experimental interfacial hydrophobicity scale derived from
partitioning studies of pentapeptides that have no secondary structure in the aqueous or
interfacial phases. This scale, that includes the peptide bonds as well as the sidechains,
can be used to calculate the virtual free energy of transfer of an unfolded chain into the
interface. The most important feature of whole-residue partitioning is that the
energetics are dominated by the peptide bonds.
2. Partitioning-Folding Coupling and the Energetics of Interfacial
Folding. A number of small peptides, such as melittin, are unfolded in the
aqueous phase, but are fully structured upon partitioning into the
interface. Even though the unfolded state is inaccessible, the energetics of
the folding can be estimated from the difference between the virtual free
energy of transfer of the unfolded state (calculated using the interfacial
hydrophobicity scale) and the measured free energy of transfer of the folded
peptide. Secondary structure formation appears to be driven by the reduction
in the free energy of partitioning of peptide bonds that accompanies
hydrogen bond formation. Experimental measurements indicate that the
reduction is about 0.5 kcal/mol per peptide bond for beta-sheet formation by
model hexapeptides and 0.4 kcal/mol per peptide bond for alpha-helix
formation by melittin . The accumulative effect of this modest reduction can
be very large (~5 kcal/mol for melittin).
3. Energetics of Bilayer Insertion. This last step in folding is the crucial one,
but the least adequately studied because of the insolubility and aggregation of
hydrophobic peptides. Direct measurement of the partitioning of a hydrophobic
alpha-helix or beta-barrel across a membrane is absolutely essential because we
must know the true cost of partitioning a hydrogen-bonded peptide bond into the
bilayer HC. Estimates for this cost vary from 0 to +1.6 kcal/mol. This means that
calculations of insertion free energy based on sidechain free energies could be
over-estimated by as much as +30 kcal/mol for a 20-residue helix!
4. Assembly of Secondary Structure Elements. The last important step is to
understand the energetics of the association of secondary structure elements
within the membrane. Don Engelman and his colleagues at Yale have shown
that transmembrane helices from bacteriorhodopsin (bR) helices that have
been independently inserted into membranes can subsequently assemble
into the native structure of bR. This indicates that the insertion steps are
independent of the intra-membrane assembly process. They refer to this
insertion-oligomerization process as the 'two-stage' model.