sfs 2

Protein tersusun dari 20 asam amino berbeda yang dihubungkan oleh ikatan peptida. Satu asam amino terdiri dari satu gugus amino, satu gugus karboksil, atom hidrogen dan satu rantai samping yang terikat pada atom karbon. Setiap asam amino memiliki rantai samping yang berbeda dan spesifik (Stansfield et al 2006). Asam amino diklasifikasikan menjadi asam amino esensial dan non esensial. Asam amino esensial adalah asam amino yang tidak mampu diproduksi sendiri oleh tubuh manusia sehingga harus dipenuhi langsung dari makanan. Asam amino non esensial merupakan asam amino yang dapat dibentuk oleh tubuh manusia dari bahan makanan yang dikonsumsi (Campbell et al 2004).
Protein memiliki empat macam struktur. Empat macam struktur itu adalah struktur primer, sekunder, tersier dan kuartener. Struktur primer protein tersusun linear sepanjang rantai polipeptida. Struktur sekunder memiliki pola pelipatan bagian – bagian polipeptida yang teratur. Pelipatan bagian – bagian antara heliks-α dan lembaran-β serta semua interaksi non kovalen menyebabkan terjadinya pelipatan yang sesuai pada suatu rantai polipeptida, peristiwa ini termasuk ke dalam struktur tersier protein. Struktur kuartener protein mengalami interaksi non kovalen yang mengikat polipeptida ke dalam satu molekul tunggal protein (Yuwono 2010).
Protein memiliki peranan penting dalam tubuh manusia. Fungsi protein dalam tubuh yaitu sebagai katalisator reksi – reaksi biokimia dalam sel, pengangkut molekul – molekul kecil dan ion, komponen sistem kekebalan tubuh, pengatur ekspresi genetik, penerus impuls saraf, serta komponen pendukung kekuatan regang (Yuwono 2010). Protein juga merupakan komponen utama dalam ribosom, sebagai hormon yang menghantarkan sinyal – sinyal kepada sel – sel yang berbeda, atau membantu pergerakan organel dalam sel (Stansfield et al 2006).
Percobaan kali ini praktikan mempelajari cara penentuan keberadaan dan konsentrasi protein. Metode yang digunakan pada percobaan ini adalah metode Bradford. Metode ini menggunakan larutan Bovin Serume Albumin (BSA) dan larutan NaCl sebagai pelarut protein. Reagen pada metode Bradford adalah Commasie Brilliant G 250. Reagen ini sering digunakan pada pendeteksian protein. Prinsip dari metode Bradford ini adalah mendeteksi keberadaan protein dengan memberikan pewarna Commasie Brilliant G 250. Semakin pekat warna ungu yang muncul maka larutan tersebut mengandung protein dan kadarnya cukup tinggi (Ahuja 2000). Selain menggunakan metode Bradford, ada beberapa metode lain yang bisa digunakan untuk mendeteksi atau menentukan kadar protein. Metode tersebut adalah metode Kjeldahl, metode Lowry, metode Dumas, metode spektrofotometri, uji biuret, uji ninhidrin, dan uji xantoproteat.

sfs

Pembahasan

Prinsip kerja autopipet adalah mengambil larutan sampel yang memiliki volume yang sangat kecil dengan menggunakan tip. Tip dipasang di ujung pipet, berfungsi sebagai penghubung antara pipet dengan larutan yang akan diambil. Tiap pipet memiliki tip yng berbeda beda tergantung dengan ukuran pipet nya. Autopipet memiliki rentang volume mulai dari 0,5 – 10 µL , 1 – 20 µL, hingga 100 -1000 µL( Balai Besar Karantina Pertanian Surabaya 2012 ). Tip yang berwarna biru cocok dipakai untuk pipet berukuran 100 -1000 µL, ukurannya lebih besar dibandingkan ukuran tip lainnya. Tip berwarna kuning cocok untuk pipet berukuran 20 – 200 µL, sedangkan untuk pipet berukuran 0,5  -10 µL menggunakan tip berwarna putih.

Tahap – tahap yang perlu diingat dalam penggunaan autopipet adalah untuk mengambil sampel praktikan cukup menekan knop atas dengan satu kali tekanan saja lalu ditahan. Praktikan lalu mengeluarkan larutan dengan cara meneka knop sebanyak dua kali dan pastikan didalam tip tidak ada  cairan yang tersisa. Untuk melepaskan tip, praktikan dapat menekan tombol yang berada di belakang knop untuk pengambilan sampel. Setelah menggunakan autopipet, praktikan harus mengatur skala volumeter pada skala maksimal autopipet tersebut. Selain itu ujung tip tidak boleh sampai tersentuh dengan tangan atau pun benda lain ( Micklos & Freyer 2003 ).

            Prinsip kerja pH meter adalah mengukur tingkat keasaman suatu larutan, selisih pengukuran pH tidak boleh melebihi 3 satuan pH antara sampel yang akan diukur dengan buffer yangdigunakan dalam pengkalibrasian pH meter. Alat ini dapat mengukur pH dengan tingkat keakuratan yang tinggi karena memiliki sensor yang canggih yaitu elektroda kaca. Elektroda ini sangat sensitif dan rapuh sehingga perlu hati – hati dalam mengoperasikan alat ini ( Day & Underwood 2001). Keakuratan yang tinggi dapat diperoleh dengan mengkalibraskan pH meter sebelum menggunakannya. Alat yang tidak dikalibrasi dapat menimbulkan kesalahan hasil analisis yang serius. Semua alat yang digunakan untuk mengukur sifat fisika senyawa harus dikalibrasi secara teratur ( Manurung 2005 ).

Ada dua metode yang bisa digunakan untuk mengkalibrasikan pH meter. Metode tersebut adalah metode kalibrasi dua nilai pH dan metode kalibrasi satu nilai pH. Pertama, elektroda dibilas dengan menggunakan air destilata lalu diseka perlahan dengan tissue yang bersih. Elektroda dicelupkan ke dalam larutan buffer yang memiliki pH 7 lalu ditunggu hingga alat ini stabil. Kemudian tombol kalibrasi diatur hingga menunjukkan angka 7 . Setelah itu elektroda dibilas lagi menggunakan air suling dan kembali diseka dengan tissue bersih lalu dicelupkan ke dalam larutan buffer pH 4. Selain larutan  buffer ber pH  4, praktikan juga dapat menggunakan larutan buffer dengan pH 10. Metode kalibrasi satu nilai pH hanya menggunakan satu larutan buffer untuk pengkalibrasian, prosedurnya masih sama dengan  metode kalibrasi dua nilai pH (  Cable 2005 ).

Praktikan menggunakan metode kalibrasi dua nilai pH untuk menggunakan pH meter. Larutan buffer yang digunakan adalah larutan buffer pH 7 dan 4.  Hasil yang diperoleh adalah sebagai berikut : pH sampel 5 yang terbaca adalah sebesar 3,63, sampel 6 memiliki Ph 3,69, dan sampel 7 memiliki pH 4. Nilai pH hamper mendekati pH buffer yang digunakan untuk kalibrasi sehingga bisa dikatakan bahwa kalibrasi yang dilakukan sudah cukup baik dan bemar.

Sentrifus merupakan alat yang memiliki prinsip kerja memisahkan sampel berdasarkan perbedaan bobot molekul sampel tersebut dengan menggunakan gaya sentrifugal. Kecepatan gaya sentrifugal ini dapat memisahkan sel dengan organel penyusunnya. Sampel dengan bobot molekul yang besar akan mengendap di dasar wadah dan disebut sebagai pelet. Sampel yang bobot molekulnya ringan akan mengapung di atasnya dan disebut supernatan. Pada fraksinasi subseluler , praktikan dapat memperoleh organel sel yang sangat kecil dengan menggunakan sentrifugasi bertingkat ( Yuwono 2010 ).

Salah satu bagian dalam alat sentrifus adalah rotor. Rotor merupakan wadah untuk meletakan sampel. Rotor tersebut dapat berputar dengan sangat cepat. Perputaran itu dapat menimbulkan panas pada sentrifus. Oleh karena itu alat sentrifus sering dilengkapi alt pendingin untuk meminimalisir panas. Panas yang berlebih dapat membuat alai ini mudah rusak. Suhu sentrifugasi berkisar 4 derajat selsius. Hal itu bertujuan untuk meminimalisir degradasi enzim yang ada terhadap komponen sel (Alberts et al 2001 ).

Larutan sampel yang praktikan gunakan pada percobaan ini adalah laritan kloroplas. Teknik sentrifugasi dapat memisahkan kloroplas dengan bagian – bagian lainnya. Namun praktikan menggunakan psentrifus berkekuatan putaran 3100 RPM dan dengan kekuatan tersebut dinilai krang cukup untuk dapat memperoleh organel yang sangat kecil. Hasil yang praktikan peroleh adalah sebagai berikut : persen rendemen untuk sampel lima sebesar 15,278  , persen rendemen untuk sampel enam 98,254, dan untuk sampel tujuh sebesar 25,742. Semakin banyak persen rendemen berarti semakin banyak pellet yang mengendap. Apabila pelet yang mengendap banyak maka supernatant yang diperoleh semakin baik dan dapat dengan mudah memperoleh organel sederhana lainnya.

 

continue

In the present analysis,
as expected, proline was found to occur predominantly in
the interfacial loop regions (see Fig. 1). Nevertheless,
unlike charged and polar (Asn, Gln) residues, it is also
represented throughout the membrane region. Indeed, it
has been suggested that prolines may increase the stability of the TM domain by “interlocking” helices, or by
providing molecular hinges that enable conformational
TABLE II. Surface Propensities of Hydrophobic and Charged Residues

Residues
Residue Propensity [%]
Surface Total
Membrane Interface Membrane Interface
Phe, Leu, Ile, Val, Ala, Gly 65.2 0.5 37.3 1.3 62.6 40.4
Phe, Leu, Ile, Val 50.4 1.0 22.4 1.1 43.2 23.8
Ala, Gly 14.8 0.6 14.9 0.2 19.4 16.6
Arg, Asp, Glu, Lys 7.3 0.6 24.1 1.4 5.8 19.0

A residue was defined to be on the surface if it has an accessibility greater than . The data was
averaged for the range 10%  50%, standard deviations are given.
Fig. 4. The hydrophobicity along the membrane normal was calculated by assigning each residue of type i a hydropathy value and
multiplying these values to the measured residue distributions ni
(z) z
along the bilayer normal. Dividing by the total number of residues in the
interval z gives the average hydropathy along the membrane normal. In
this figure all residues with a surface accessibility of x  20% were
considered to be on the surface.
DERIVATION OF AN IMPLICIT MEMBRANE POTENTIAL 257transitions.
93,94
Glycine was found to have a preference for
the membrane region, behaving more like a hydrophobic
residue. This preference was not detected in our previous
analysis with a reduced dataset but is consistent with
other studies which found Gly to be twice as abundant in
membrane proteins than soluble proteins.
36,73
Potentials of Mean Force
Figure 5 shows the fitting of smooth Gaussian functions
to the normalized distributions after subtraction of the
reference state (i.e., division by the total distribution, see
Methods). All four different types of topology that were
used in the fitting are shown by a representative residue
(Arg, Leu, Gln, and Trp). Hydrophobic residues Ala, Ile,
Leu, Val as well as Phe, Gly, and Met were fitted with a
single upright Gaussian. Polar residues Asn, Gln, and Pro
were fitted with a single inverted Gaussian centered in the
membrane. Aromatics Trp, Tyr, and His were fitted with
two upright Gaussians one at each membrane interface.
Charged residues Arg, Asp, Glu, and Lys were fitted with
double Gaussians, one inverted near the membrane center
and another upright at the cytoplasmic interface. Residues
Cys, Ser, and Thr were not fitted. Cys because it occurs too
infrequently to be statistically valid and Ser and Thr
because the potential is essentially flat after subtraction of
the reference state.
Figure 6 shows the corresponding potentials of mean force
for all residues. Table I lists the fitting parameters, 
2
error
values as well as the correlation coefficients and RMS errors
for all amino acids. In general, the quality of the fit is very
good, with hydrophobic and charged residues displaying the
best correlations. These results are encouraging and demonstrate that the use of Gaussians is a reasonable approximation. It should be noted that curves were only fitted in the
range 45 to 45 Å since beyond this range the number of
residues drops significantly (c.f. Fig. 1).
Hydrophobic residues display a potential energy well
near the center of the membrane region (see parameter a3
in Table I) and extending into the interfacial regions [Fig.
6(B)]. This agrees with mass spectrometry experiments on
synthetic membrane peptides that found that introducing
Ala and Leu residues into the polar interfacial regions
seems to have relatively small energy penalties.
87
The free
energy of transfer from water to the membrane interface
can be compared with the experimental interface scale of
Fig. 5. The measured frequencies (solid lines) and fitted functions
(dashed lines) for the four different types of functions fitted.
Fig. 6. Potentials of mean force for charged (A), hydrophobic (B),
polar (C) and aromatic residues (D). The energy is given in kT  0.6
kcal/mol at T  300 K.
258 M.B. ULMSCHNEIDER ET AL.Wimley and White.
83
They found values of 0.31 0.06
kcal/mol and 0.56 0.04 kcal/mol for Ile and Leu
respectively, which compare to our values of 0.25 0.02
kcal/mol and 0.28 0.02 kcal/mol, obtained by averaging
over both membrane interfaces.
For charged residues, the potentials of mean force have
a narrow peak at the membrane center and a slight
depression at the cytoplasmic interface [Fig. 6(A)]. The
cost of burying a charged residue within the hydrocarbon
core of a lipid bilayer is extremely high (9 kcal/mol for a
Lys residue
15
). However, in the current potentials it is only
3 kcal/mol, much smaller than the theoretical cost of
neutralization and burial of 10 –20 kcal/mol.
44
This discrepancy can largely be attributed to the extremely low propensities of charged residues in the membrane center, making
good fitting in this region very problematic (a zero residue
propensity results in an infinite potential, while small
changes close to zero produce large changes in the potential). Thus the current method clearly underestimates the
penalty for charged residues to be buried in the membrane
center.
It should also be noted that the present analysis cannot
distinguish between charged and neutral residues. One
third of ionizable residues at the membrane interfaces
have surface accessibilities greater than 50% and are
therefore almost certainly charged. Thus the average free
energy of transfer from water to the membrane interface
for Asp and Glu (assuming one-third charged and twothirds neutral) according to Wimley and White
83
is 0.36
0.10 kcal/mol and 0.66 0.14 kcal/mol respectively. This
compares to our values of 0.39 0.04 kcal/mol and 0.41
0.03 kcal/mol respectively, which were averaged over both
bilayer interfaces.
Experimental evidence suggests that, while resisting
partitioning into the membrane below the level of the
phosphates, Lys does not appear to resist displacement
from the interface towards the aqueous phase,
54
in good
topological agreement with the shape of the present potentials.
Aromatic residues (His, Trp and Tyr) have potentials of
mean force with two wells, one at each membrane interface [Fig. 6(D)]. This potential shape was expected from
structural, experimental, and computational data (see
above). The penalty of moving Trp or Tyr from the interfaces to the aqueous domain was found to be 0.68 kcal/mol
and 0.47 kcal/mol respectively, much lower than the
corresponding values from Wimley and White’s interface
scale (1.85 kcal/mol and 0.94 kcal/mol).
Polar residue potentials (only Asn and Gln, see above)
display a single broad peak centered in the membrane
[Fig. 6(C)]. The energy penalty of displacing a polar
residue from the solvent to the interface is relatively small
0.1 kcal/mol, while the penalty for insertion into the
membrane core is around 2 kcal/mol.
Generally, topological differences within each group of
residues (i.e., hydrophobic, charged, aromatic, and polar)
are small and show only subtle differences in the distributions and resulting potentials of mean force. This agrees
well with experimental observation from synthetic transmembrane peptides which found only minor differences on
substitution of Lys with Arg and Trp with Tyr as flanking
residues.
64,65
Finally, it should be noted that the energies of transfer
from the interface to the aqueous solution, although
slightly different in magnitude (average error of 0.5 kcal/
mol for the interface scale and 1.0 kcal/mol for the octanol
scale), nevertheless correlate highly with both the octanol
(85%) and interface scale (88%) of Wimley and White.
83
This correlation is highest for the hydrophobic (98%),
charged, and polar residues (80%), while there is little
correlation for aromatics (33%). For insertion into the
center of the membrane the free-energy correlations are
87% with the octanol scale and 78% with the interface
scale. This means that the present interface free energies
correlate better with the experimental interface scale,
while the buried scale correlates better with the experimental octanol scale, which is encouraging for the correctness
of the overall shape of the potentials.
However, it should be noted that the current study is not
attempting to provide accurate free-energy profiles but to
make an initial assessment of the validity of using transmembrane residue distributions to derive an implicit
membrane representation for simulation studies.
Membrane Protein Insertion
The potentials of mean force were tested on various
membrane proteins: bacteriorhodopsin (1cwq.pdb),
95
sensory rhodopsin (1h68.pdb),
96
the KcsA potassium ionchannel (1k4c.pdb),
97
the GLPT glycerol-3-phosphate transporter (1pw4.pdb),
98
the glycophorin A dimer (1afo.pdb),
99
as well as two aquaporins (1j4n.pdb and 1rc2.pdb)
100
and
two chloride channels (1kpk.pdb and 1kpl.pdb).
101
Residue
distributions were calculated leaving each protein out in
turn. However, the deviations between the distributions
were found to be much smaller than the error in the curve
fitting (see Methods). For the distribution without GLTP,
the largest protein in the test set, the error with respect to
the total distribution was 
2
 1.4  10
5
, resulting in
identical curve fits. The errors for the other proteins are
even smaller.
The aligned proteins were moved through the membrane and the energy recorded as a function of the distance
from the membrane center. Figure 7 demonstrates that
the completely inserted configuration is at an energy
minimum for all membrane proteins investigated (c.f. zmin
in Table III). The energy minima were found to be within
2.5 Å of the membrane center.
All energy profiles are asymmetric across the membrane. Insertion from the cytoplasmic side is more favorable, exhibiting no energy barrier, while the extracellular
side has a steeper gradient and (with the exception of
sensory rhodopsin and one aquaporin) shows a slight
penalty for insertion. This result agrees well with the
solvation energy profile recorded for a recently developed
generalized Born implicit membrane representation,
49
which also found insertion from the cytoplasmic region
more favorable. However, their energy of solvation was
found to be much higher being 143.1 kcal/mol for bacterioDERIVATION OF AN IMPLICIT MEMBRANE POTENTIAL 259rhodopsin, compared to the 42.9 kcal/mol found in this
study.
In the inserted configuration the membrane model was
tested by rotating the protein in the center of the membrane. The minimal tilt angles are given in Table III and
are in the range 0°–15°, except for the aquaporins which
have tilt angles nearer 30°. All proteins have a second
minimum near 180° (i.e., upside down in the membrane),
but in all cases this was found to have significantly higher
energies (20%) suggesting that the present potential
captures the inside/outside orientation of the proteins
correctly. For bacteriorhodopsin the tilt angle of 13° compares well with the 12° from the crystal structures.
Interestingly the insertion profiles of proteins with more
irregular secondary structures like aquaporins and chloride channels do not differ from the those of very regular
structures such as bacteriorhodopsin or KcsA (c.f., Fig. 7).
Trans-Membrane Helices
Figure 8 shows the energy profiles for helices A and C of
bacteriorhodopsin (from 1cwq.pdb) as well as monomeric
and dimeric glycophorin A (from 1afo.pdb). Two types of
orientations were investigated, parallel to the membrane
normal and parallel to the membrane surface.
A stable helical trans-membrane configuration has been
experimentally verified for helix A,
102
and is well docuTABLE III. Energy of Insertion into the Membrane for the
Glycophorin A Dimer (GpA 2x), Bacteriorhodopsin (BR),
Sensory Rhodopsin (SR), the KcsA Potassium Channel, the
Glycerol-3- Phosphate Transporter (GLPT), Aquaporins
fromBovine red blood cell(AQPI) and E. coli(AQPZ) as
Well as Chloride Channels fromE. coli(CIC 1)
and S. typhimurium(CIC 2)

Protein
Energy minima
Emin
[kcal/mol] zmin
[Å] min
[degrees]
GpA 2x 23.2 1.5 4
BR 42.9 0.0 13
SR 46.7 1.5 2
KcsA 90.3 0.5 1
GLPT 88.9 0.0 15
AQPI 46.0 1.0 25
AQPZ 52.1 1.0 28
CIC 1 92.0 2.5 13
CIC 2 90.4 2.5 0

The depth of the energy well Emin
, optimal tilt angle min
, and
position with respect to the membrane center zmin
are given.
Fig. 7. Insertion energy profiles derived by pushing the aligned
proteins through the potential of mean force membrane representation.
The extracellular side of the membrane is to the left (negative z-axis). The
proteins shown are bacteriorhodopsin (BR), sensory rhodopsin (SR), the
KcsA potassium channel, the glycerol-3-phosphate transporter (GLPT),
aquaporins from Bovine red blood cell (AQP1) and E. coli (AQPZ) as well
as chloride channels from E. coli (ClC 1) and S. typhimurium (ClC 2).
Proteins shown in B have a more irregular secondary structure than those
shown in A.
Fig. 8. Helices A and B of bacteriorhodopsin as well as monomeric
(GpA) and dimeric (GpA 2x) glycophorin A. A: the energy profile of
inserting the helix perpendicular to the membrane surface. B: the profile
for a helix parallel to the membrane surface.
260 M.B. ULMSCHNEIDER ET AL.mented for glycophorin A. Helix C on the other hand is one
of the few systems for which quantitative binding and
insertion data is available.
63
At neutral pH, it associates
with the membrane in a nonhelical probably peripheral
conformation, while forming a stable TM helix upon
protonation of its aspartate residues.
103
The current study found that all helices aligned parallel
to the membrane normal have an energy minimum close to
the membrane center. The relative energy differences with
respect to the aqueous domain are 7.8 kcal/mol, 4.4
kcal/mol and 11.5 kcal/mol for helix A, C, and the
glycophorin A monomer respectively. These values compare well with experimental estimates of the free energy of
insertion for a single TM helix, which are in the range of
5–12 kcal/mol.
103–106
It should be noted, however, that
experimental difficulties make these values somewhat
unreliable.
63,107
Both the monomeric and dimeric glycophorin A exhibit a
slight penalty for crossing into the extracellular space. The
dimer has exactly twice the insertion energy (c.f., Table
III) compared to the monomer at a tilt angle of 4° compared
to 27° for the monomer. The insertion energy for the dimer
is comparable to values obtained from PB/SA calculations
(18 kcal/mol).
44
Moving the helices across the membrane while keeping
their axes parallel to the membrane surface showed a very
interesting feature of the present potentials. All helices
exhibit potential energy wells close to the interfacial
regions [ 10 –13 Å, see Fig. 8(B)]. At the center of the
membrane, conformations perpendicular to the membrane
normal have significantly higher energies than the TM
configurations. This is not necessarily the case near the
interfaces. In fact, helices A and C have 2– 4 kcal/mol lower
energies when oriented parallel to the membrane surface
at the intracellular interface. Glycophorin A behaves
similar at the extracellular interface. The surface parallel
interfacial configuration of helix C was even found to be
lower than the inserted configurations, which is a remarkable finding since experimental evidence indeed suggests a
partially unfolded surface bound conformation.
103
Incidentally a recently developed energy function for membrane
peptides and proteins also found a partially unfolded
interfacial configuration to have lower energies than the
TM configuration.
44
The calculations were repeated for the ten NMR structures of the M2 helix of the -subunit of the actylcholine
receptor.
108
The curves are exactly similar to those in
Figure 8 (data not shown). Generally inserted TM configurations are the most stable, with an average energy
minimum of 4.6 0.1 kcal/mol at the center of the
membrane (0.9 0.6 Å) and the optimal tilt angle of 9
5° is comparable to the 12° determined by NMR.
108
Adsorption of the peptide onto the membrane surface is
also favorable but to a lesser extent, with energy minima of
3.0 0.6 kcal/mol for the cytoplasmic (9.4 0.6 Å) and
2.4 0.7 kcal/mol for the extracellular interface (11.0
1.0 Å).
These results are in excellent agreement with a recent
theoretical study of the same structures,
109,110
which
found average energies of 4.7 2.1 kcal/mol and 2.6
2.4 kcal/mol for inserted and surface bound configurations
respectively. The study used a theoretical continuumsolvent method developed by Ben-Tal
111
that has been
successfully applied to estimate the insertion energies of
TM peptides and proteins.
112
In order to compare the
results the helix– coil transition free energy (Gcon  2.4
kcal/mol) was subtracted, since the present data estimates
the insertion energy of a folded helix.
Future Improvements
Future improvements of the present membrane representation might have to include separate potentials for surfaceaccessible ionizable residues. Also, the surface dependence
of the hydropathy analysis suggests that the strictly
additive nature of the potentials (c.f., GpA monomer/
dimer) might be overestimating the free energy of membrane insertion of proteins with larger trans-membrane
segments. It should be noted that the current potentials
were derived from folded conformations only. Therefore it
is not entirely certain that the resulting potentials are
sufficient to study protein folding or if a free-energy term
associated with backbone exposure has to be included.
Distributions at the Protein Surface
Figure 9 shows the correlation of the total and surface
distributions as a function of the residue accessibility. A
residue accessibility of 10% means that a residue with
more than 10% of its side chain surface area accessible to
the environment (solvent or membrane) is considered a
surface residue. The figure demonstrates that there is a
very strong correlation for all but the most exposed
hydrophobic residues. Less than a quarter (23%) of resiFig. 9. Correlation of surface and overall distributions according to
residue types (charged, hydrophobic, polar, aromatic, and total). The
number of residues located at the surface as a fraction of all residues is
also shown (surface fraction). The correlations are plotted against the
fraction of a side chain that has to be accessible in order for that residue to
be considered on the surface of the protein (accessibility fraction). For
residues with up to 50% of their side chains exposed to the environment
(membrane or water) the correlation is over 90%, even though they
represent just 25% of all residues.
DERIVATION OF AN IMPLICIT MEMBRANE POTENTIAL 261dues have side-chain accessibilities greater than 50%.
Nevertheless their correlation with the total distribution
is still over 90%. Interestingly the correlation is highest for
charged (95%) and hydrophobic (95%) amino acids.
Current theory states that -helical membrane proteins
fold by forming and inserting their helices individually or
in pairs and assembling them at a later stage.
52,113
Indeed
individual fragments of bacteriorhodopsin form secondary
structure when immersed in a membrane environment
and subsequently combine to form a functional protein.
114,115
Consequently all TM segments, whether buried
inside the protein or exposed to the lipid bilayer, should
exhibit the same distribution pattern, since they insert on
their own. This is in excellent agreement with present
results (c.f., Fig. 9).
In the present study the membrane potential was derived from the distributions of all residues, which is
justified for -helical membrane proteins by the above
analysis. However, residues with very high surface accessibilities probably contribute more to the insertion free
energy than buried residues. On the other hand many
biological bilayers (such as the mitochondrial or purple
membranes) have extremely high protein densities, leading to significant protein–protein contacts. Furthermore,
many membrane proteins, even in membranes of lower
protein lipid ratios are oligomers. As a result it is difficult
to estimate exactly how much of a residue is exposed to the
solvent or membrane environment, and consequently the
surface accessibility contribution to the free energy of
insertion is difficult to assess. Nevertheless, the current
analysis seems to indicate that the insertion of a protein
fragment into a membrane might be energetically similar
to its burial inside a membrane protein.
CONCLUSION
It is generally recognized that overall hydrophobicity is
the main driving force for the integration of -helical
trans-membrane segments into the lipid bilayer.
116
The
current study found that the vast majority of residues in
the membrane domain are hydrophobic. Furthermore, the
protein surface facing the lipids was found to be even more
hydrophobic than the protein core, suggesting that membrane proteins can indeed be regarded as somewhat
“inside-out,” at least regarding their membrane domains.
The distributions of all amino acids were found to be
symmetric with the exception of the four charged residues,
which occur more frequently on the cytoplasmic side of the
membrane. In addition to this asymmetry they were found
to be distributed such as to cause a net charge imbalance
across the membrane domain, in line with the positive
inside rule.
The variation within each group of residue distributions
(i.e., hydrophobic, charged, aromatic, and polar) were
found to be small and caused only subtle differences in the
resulting potentials of mean force. The shape of the
potentials were shown to be consistent with experimental
data and correlate well with measured free energies of
solvation both for buried and interfacial locations.
The resulting membrane potential was tested on several
integral membrane proteins. In all cases the correctly
inserted orientation was found to be at a clear energy
minimum. Further investigations with single transmembrane -helices found that both inserted and surface
bound conformations are at energy minima, consistent
with theoretical, experimental, and simulation data.
The translational and rotational energy profiles described here represents a fairly limited search of the
orientation space of the peptides and proteins considered.
Nevertheless the present preliminary study has clearly
demonstrated that the number of membrane proteins
solved at atomic resolution is now sufficient for a detailed
statistical analysis of the amino acid distribution functions
as well as the derivation of meaningful potentials of mean
force. The smoothness of the energy profiles is remarkable
and the good overall agreement with experimental, statistical, and simulation data is encouraging.
ACKNOWLEDGMENTS
MBU is funded by the Wellcome Trust. Research in
MSPS’s group is supported by the Wellcome Trust. We
would like to thank Saraswathi Vishveshwara and the
Molecular Biophysics Unit at the IISc, Bangalore, India for
providing facilities and Phil Biggin for his comments on
this manuscript.
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264 M.B. ULMSCHNEIDER ET AL.APPENDIX. List of Membrane Proteins Used in the Current Study
Protein, Organism Resolution [Å] PDB Date
Light active proteins
Bacteriorhodopsin, H. salinarium 1.6 IC3W 1999
Halorhodopsin, H. salinarium 1.8 1E12 2000
Sensory rhodopsin II, N. pharaonis 2.1 1H68 2001
Rhodopsin, Bovine rod outer segment 2.8 1F88 2000
Photosynthetic reaction center, R. virdis 2.3 1PRC 1985
Photosynthetic reaction center, R. sphaeroides (Replace 4RCR) 2.4 IOGV 2003
Photosynthetic reaction center, T. tepidum 2.2 IEYS 2000
Light harvesting complex, R. acidophila 2.0 INKZ 2003
Light harvesting complex, R. molischianum 2.4 ILGH 1996
Photosystem I, S. elongates 2.5 IJB0 2001
Photosystem II, T. vulcanus 3.7 IIZL 2003
Cytochrome b6f complex,M. laminosus 3.0 IUM3 2003
Cytochrome b6f complex, C. reinhardtii 3.1 IQ90 2003
Channels
KcsA potassium channel, S. lividans 2.0 IK4C 2001
MthK potassium channel,M. thermoautotrophicum 3.3 ILNQ 2002
KirBac1.1 Inward-Rectifier potassium channel, B. pseudomallei 3.7 IP7B 2003
MscL mechanosensitive channel,M. tuberculosis 3.5 IMSL 1998
MscS voltage-modulated mechanosensitive channel, E. coli 3.9 IMXM 2003
CIC chloride channel, S. typhimurium 3.0 IKPL 2002
CIC chloride channel, E. coli 3.5 IKPK 2002
Acetylcholine Receptor Pore, T. marmorata 4.0 IOED 2003
AQP1-aquaporin water channel, Human red blood cell 3.7 IIH5 2001
AQP1-aquaporin water channel, Bovine red blood cell 2.2 IJ4N 2001
AQPZ-aquaporin water channel, E. coli 2.5 IRC2 2003
SecYE -protein-conducting channel,M. jannaschii 3.5 IRHZ 2003
GlpF-glycerol facilitator channel, E. coli 2.2 IFX8 2000
Transporters
AcrB-bacterial multi-drug efflux transporter, E. coli 3.5 IIWG 2002
LacY-lactose permease transphate transporter, E. coli 3.5 IPV7 2003
GlpT-glycerol-3-phosphate transporter, E. coli 3.3 IPW4 2003
BtuCD-vitamin B12
transporter, E. coli 3.2 IL7V 2002
Respiratory Proteins
Fumerate reductase, E. coli 3.3 IL0V 1999
Fumerate reductase,W. succinogenes 2.2 IQLA 1999
Calcium ATPase, Rabbit sarcoplasmic reticulum 2.6 1EUL 2000
F1
F0
ATP Synthase–H Transporter C subunit, E. coli NMR 1A91 1998
F1
F0
ATP Synthase–B subunit, E. coli NMR IB9U 1999
Formate dehydrogenase-N, E. coli 1.6 IKQF 2002
Succinate dehydrogenase (Complex II), E. coli 2.6 INEK 2003
NarGHI Nitrate reductase A, E. coli 1.9 IQ16 2003
Mitochondrial ADP/ATP Carrier, Bovine heart mitochondria 2.2 IOKC 2003
Cytochrome C oxidase (aa3
), Bovine heart mitochondria 2.8 IOCC 1996
Cytochrome C oxidase (aa3
), P. denitrificans 2.8 IARI 1995
Cytochrome C oxidase (ba3
), T. thermophilus 2.4 IEHK 2000
Cytochrome bc1
complex, Bovine heart mitochondria 3.0 IBGY 1998
Cytochrome bc1
complex, Chicken heart mitochondria 3.2 IBCC 1998
Cytochrome bc1
complex, S. cerevisiae 2.3 IEZV 2000
Glycophorin A. Human red blood cell NMR IAFO 19

protein integral

Properties of Integral Membrane Protein Structures:
Derivation of an Implicit Membrane Potential
Martin B. Ulmschneider,
1
* Mark S.P. Sansom,
2
and Alfredo Di Nola
1
1
Department of Chemistry, University of Rome ‘La Sapienza,’ Roma, Italy
2
Department of Biochemistry, University of Oxford, Oxford, United Kingdom
ABSTRACT Distributions of each amino acid
in the trans-membrane domain were calculated as a
function of the membrane normal using all currently available -helical membrane protein structures with resolutions better than 4 Å. The results
were compared with previous sequence- and structure-based analyses. Calculation of the average hydrophobicity along the membrane normal demonstrated that the protein surface in the membrane
domain is in fact much more hydrophobic than the
protein core. While hydrophobic residues dominate
the membrane domain, the interfacial regions of
membrane proteins were found to be abundant in
the small residues glycine, alanine, and serine, consistent with previous studies on membrane protein
packing. Charged residues displayed nonsymmetric
distributions with a preference for the intracellular
interface. This effect was more prominent for Arg
and Lys resulting in a direct confirmation of the
positive inside rule. Potentials of mean force along
the membrane normal were derived for each amino
acid by fitting Gaussian functions to the residue
distributions. The individual potentials agree well
with experimental and theoretical considerations.
The resulting implicit membrane potential was
tested on various membrane proteins as well as
single trans-membrane -helices. All membrane proteins were found to be at an energy minimum when
correctly inserted into the membrane. For -helices
both interfacial (i.e. surface bound) and inserted
configurations were found to correspond to energy
minima. The results demonstrate that the use of
trans-membrane amino acid distributions to derive
an implicit membrane representation yields meaningful residue potentials. Proteins 2005;59:252–265.
© 2005 Wiley-Liss, Inc.
Key words: amino acid distribution; membrane protein; implicit membrane; potential of
mean force; -helices
INTRODUCTION
Integral membrane proteins play a crucial role in cell
function and communication. Current estimates indicate
that 20 –30% of the human genome encodes membrane
proteins.
1–3
Even though the majority of drug targets are
membrane proteins such as receptors and ion-channels
4
only 46 high-resolution structures of different membrane
proteins are known at present. The scarcity of structural
data is mainly a result of substantial difficulties with
over-expression and crystallization of membrane proteins.
5
Recently, promising developments in the methodology of membrane protein structure determination have
been reported.
6–9
Nevertheless it seems unlikely that the
rate of structure determination will increase significantly
in the near future.
The relative paucity of structural data has impeded the
development of knowledge-based potentials that have
been successfully applied in globular protein structure
prediction.
10
Instead, a set of methods with increasing
levels of sophistication has been developed to predict the
topology of trans-membrane (TM) -helices in membrane
protein sequences, reaching accuracies close to 100%.
11–13
The prediction methods can be divided into two broad
classes: i) hydrophobicity analyses of membrane protein
sequences based on theoretical or experimental physiochemical considerations
13–18
and ii) statistical analyses
based on known membrane protein structures or databases of experimentally confirmed membrane protein topologies.
19 –26
These methods have been employed to analyse the
residue distributions and general properties of TM helices
27,28
and membrane protein structures
29 –31
in order to
extract common features. Others have concentrated on the
role of individual residues such as proline-induced kinking
of TM helices
32
or the importance of glycine in TM helix
association.
33
Analyses of residue distributions have also
been used to study the packing of membrane proteins
34 –36
and to derive knowledge-based scales for membrane protein prediction and folding.
37,38
The present work can be divided into two parts. The first
is a detailed analysis of the distributions and preferred
locations of each amino acid in the membrane domain
using all currently available -helical membrane protein
structures. This analysis closely follows a previous publication,
29
which suffered from the scarcity of structures
available at the time. Recent years, however, have seen a
considerable increase in the number of membrane protein
Grant sponsor: Wellcome Trust
*Correspondence to: Martin B. Ulmschneider, Department of Chemistry, University of Rome ‘La Sapienza,’ Piazzale Aldo Moro 5, I-00185
Roma, Italy
Received 22 July 2004; Accepted 6 August 2004
Published online 18 February 2005 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/prot.20334
PROTEINS: Structure, Function, and Bioinformatics 59:252–265 (2005)
© 2005 WILEY-LISS, INC.

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