Ingeniera Bioqumica!

(1) Escoge la respuesta correcta:

4. Cul de las siguientes fuerzas es la ms

favorable para el plegamiento de las protenas?
A. entropa conformacional
B. Interacciones hidrofbicas
C. interacciones Vander Waals
D. Los enlaces de hidrgeno
5. En el punto medio de una curva de transicin de
temperatura,
A. la mitad de la estructura molcular de la protena
est desnaturalizada
B. Keq = 1,0 y G = 0
C. [Nativa] = [Desplegado]
D. Todas las respuestas son acertadas

curso 2015/2016

10. Si la protena de clara de huevo, ovoalbmina,

se desnaturaliza en un huevo duro, cul de los
siguientes es la menos afectada?
A. La estructura primaria de la ovoalbmina
B. La estructura secundaria de la ovoalbmina
C. La estructura terciaria de la ovoalbmina
D. La estructura cuaternaria de la ovoalbmina

(2) En la figura se muestra el incremento de energa

libre de estabilizacin (G=H-TS) de
diferentes protenas: una mesoflica y tres
termoflicas.
Qu representan los mnimos (puntos redondos
huecos) de los perfiles?
Podras indicar qu representan los puntos de corte
(cuadrados) con el eje de las temperaturas?
Qu protena/s son meso y termoflicas?
Qu conclusiones generales extraes sobre las
diferencias entre enzimas meso y termoflicas? 184
reviews
Justifica todas
respuestas
razonadamente.
W.F. Li et tus
al. / Biotechnology
Advances
23 (2005) 271281

6. Cul de las siguientes es la ms correcta?

A. los aminocidos cargados nunca estn enterrados
en el interior de una protena
276
B. aminocidos cargados rara vez son enterrados en
16,18). The
of 0.2 M K+ ions to wild enzyme resulted in a 10 8C increase in the thermal denaturation
el interior de una protena
AG slab
mesozymes a
temperature. No significant increase
was observed in mutants E98D and E98S, indicating
1
C. Todos los aminocidos hidrofbicos se
accumulation
that the mutation of Glu98 eliminates cation binding (Radfar et al., 2000).
hydrogen bon
encuentran enterrados cuando una protena se
actions. Owing
tures, the im
pliega
4. Good conformational structure
nations for a
D. la tirosina slo se encuentra en el interior de las
universal prot
4.1. More rigid, less flexible
rules for propo
protenas
mesozymes

8. Ya que G =-RTlnK
A. un aumento de 10 veces en K disminuye G en
aproximadamente 10-veces
B. una disminucin de 10 veces en K disminuye G
en aproximadamente 2,3 * RT
C. un aumento de 10 veces en K disminuye G en
aproximadamente 2,3 * RT
D. una disminucin de 10 veces en K aumenta G
en aproximadamente 10-veces

increased
rigi
The free energy of stabilization
(AGstab) of globular
catalytic active
mesozymes is typically 5-15 kcalmolli
at 25C (Ref.
unfolding.
Su
50
12). Most thermozymes
studiedAdenylate
originatekinase
fromfrom
therhydrogen-exchang
mophiles, but the AC&, values of only
a few thermoto proteolytic
S. acidocaldarius
zymes have been determined:
at 25C
AC&
denaturant
u
porcine the
cytosol
40
values of Tkermus tkermopkilus
cytochrome
c-552
amino acid su
and phosphoglycerate
kinase were shown to be only
thermostability,
30
15 kcalmol-1
and 3-6.5 kcal mol-l higher
than the
enzymes
ind
counterparts,
respecto improve
AGEtab values of their mesophilic
20
tively..
Stability studies on mutant enzymesis~
through cavity
show that AAG,,,, (i.e. changes in the AGstab) values as phobicity),
a
small as 3-6.5 kcalmol-i
can account for thermoity (e.g. throug
10
stability increases of up to 12C. These data confirm
interaction
o
that the difference in stabilization
energies of thermoreduction).
0
zymes and mesozymes is small, and is usually in the
range 5-15
of thermoHydrophobic i
40 kcalmol-l. 60The AGstab values
80
20
100
zymes originatingTemperature
from hyperthermophiles
have not
Hydrophobic
(C)
been determined.
Predictions
for Pyrococcus furiosus
energy required
a-glucosidase
indicate andthat
theymuscle
mightcytosol
be adenylate
signifitions. during
From
Fig. 1. Hydrogendeuterium exchange
in S. acidocaldarius
porcine
kinases
cantly
higher
than the protons
corresponding
values were
tations
in four
a temperature gradient experiment.
Fractions
of unexchanged
as a functionAGstab
of temperature
calculated
!1
from the normalized amide II intensities
1546 (S. acidocaldarius
and 1542 cm
enzyme).
of their at mesophilic
counterparts.enzyme)
In addition,
if the(porcine
buried
-CH,
From Bonisch et al., 1996.
temperature
for maximum
stability of a thermozyme
the conformation
is different from that of its mesozyme counterpart,
the
that hydrophobic
difference in their AG,,, values at 25C is meaningphilicity and
less (Fig. 1). For relevant comparisons,
more infor-

Unexchanged protons (%)

7. Cul de las siguientes fuerzas es la ms

desfavorable para el plegamiento de las
protenas?
A. entropa conformacional
B. interacciones hidrofbicas
C. interacciones Van der Waals
D. interacciones electrostticas

analysesi)is ha
Thermozymes are generally more rigid than mesoenzymes. This increased rigidity
Figure 1
thermozyme
essential for preserving their catalytically
active structure at elevated temperature.
Comparison of theoretical mesozyme and thermozyme free energy of stabilization
thermozyme
Increased rigidity
a protein
unfolding.
Overall
rigidity
increases
(AG,,,)protects
curves. (a)
Theoreticalagainst
AGStabcurve
for a mesozyme
with enzyme
a temperature
of
different mech
through a-helix
stabilization,
optimization,
maximal
stability (J,;electrostatic-interaction
open circle) of 20C and a melting
temperature conformational-strain
(J,; open
Most molec
(3) etc.
En
unofexperimento
securves
sita
una
protena
en una
reduction,
Enhanced
rigidity
is AG,,,,
demonstrated
by reduced
square)
50C. (b-d)
Theoretical
for a thermozyme
with a J, hydrogendeuterium
of 100C:
stabilization
the thermozyme
the same
J, as the
mesozyme
-the thermozymes
curve is denaturants,
exchange
rates,fb)lower
susceptibility
to proteolytic
degradation
and chemical
studies. Becaus
disolucin
quehascontiene
agua
deuterada
a distintas
downward;
(c) theunfolding.
thermozyme Fig.
and 1
mesozyme
have data
different
crystallized,
c
and reduced shifted
thermally
induced
illustrates
of J,a hydrogen
temperaturas.
un
values, but the sameTranscurrido
AG,,,, at their respective J,
- thedeterminado
thermozymes curve is shifted
structures
vali
deuterium exchange
experiment.
At
20
8C
a
much
smaller
fraction
(53%)
of
the
amide
towards higher temperatures; (d) the thermozyme and mesozyme with the same J,
posed by
se
protena
y
se determina
la kinase
protonstiempo
in the
relatively
Sulfolobus
acidocaldarius
adenylate
areearli
values
and extrae
the samestable
AGla
stabvalues
at J, -the thermozymes
AG,,,, curve
is flatter.
structural com
exchanged
than
in
the
less
stable
porcine
cytosolic
enzyme
(83%).
The
exchange
was
AG,,,
values
are
usually
compared
at
25C.
If
one
of
the
enzymes
has
a
J,
far
from
proporcin de deuterio que ha sido incorporado protein
a
stabiliz
(c)l, the
a comparison
their S.
AGstab
values at 25C is meaningless.
complete at 5625C
and[e.g.
97enzyme
8C for
porcineofand
acidocaldarius
enzymes, respectively
los
grupos
amida
de
la
protena
Cmo
podras
Athorough
comparison
should
include
the
J,
and
J,
of
each
enzyme,
and
their
AG,
(Bonisch et al., 1996).
What makes
values at their respective J,. Data accumulated on Jhermus thermophilus phosphoThe explicar
double
mutant resultados
K18G/R82E of
BacTrx
(Alicyclobacillus
acidocaldarius
deindicate
la grfica
de abajo
Thermozyme
glyceratelos
kinase and cytochrome c-552
that the dependence
of the AG,,,
thioredoxin) had
reduced heat
resistance
comparedandtothat
theit involves
thermostable
wildtype
protein.
Because the
on temperature
is unique
to each thermozyme,
one or more
of
sabiendo
que
se
estn
ensayando
una
protena
more were
severely
Protein dynamics
analysisshown
shows
that both the mutant and wildtype protein
the mechanisms
aboveW4.
need to
termoflica?
Identifcalas.
globallymeso
as rigidyasotra
the mesophilic
thioredoxins
at room temperature, but the they
wildtype

Ingeniera Bioqumica!

curso 2015/2016

(4) Qu podras decir acerca de estas cuatro

enzimas catalasa de diferente origen sabiendo que
se ha medido su desnaturalizacin (mediante su
actividad cataltica) a diferentes tiempos en
idnticas condiciones (50C) y tras ser
reconstituidas en un tampn adecuado? Haz una
estimacin de la vida media de cada una de ellas.
Proc. Natl. Acad. Sci. USA 95 (1998)

nal model of the

is shown in blue.
ated in this study
n in red and the
he critical region
shown in yellow.
n.

FIG. 2.

2057

First-order inactivation of TLPs. Stabilities of TLPs were

determined at different temperatures. Shown are the first-order

(5) La creciente
comprensin de cmo se pliegan
inactivation curves for TLP-ste incubated at 80C () and 90C ("),
thermolysin at 90C (), and the 8-fold mutant at 100C (F).
las protenas ha permitido a los investigadores
suringpredicciones
the change in absorbance
at 345
change in
establecer
acerca
denm.laThe
estructura
absorbance was linear during the time of measurement (3060
nyl-L-phenylalamin),
indicating that en
the los
proteases
were de
active
this time
proteica
basndose
datos
lainsecuencia
m ratios of the
interval and that substrate depletion was negligible.
yme concentraprimariaProteolytic
de aminocidos.
Responde
a incubated
las
(1 mg!ml) was
Properties. #-Casein
st-order condiwith TLP-ste or the 8-fold mutant at a molar ratio of 1,000:1
lly according
to
siguientes
una
manera
justificada.
for 1 cuestiones
hr at 60C and de
100C,
respectively.
At both
these
or phosphoramtemperatures, #-casein behaves as a noncompact and largely
(a)L-Basndose
en
la
secuencia
de
aminocidos
de la
phosphinyl]flexible structure (23). The peptides resulting from hydrolysis
min preincubawere
derivatized
with
dansyl-chloride.
The
proteolytic
prodfigura; dnde se puede predecir que no se
concentrations
ucts were separated by loading a sample corresponding to 50
MOPS, pH 7.0!5
"g #-casein
on a reversed-phase column (RP-304, Bio-Rad).
producirn
-hlices?
of the furylacThe mobile phase used was 50 mM NaAc, pH 5.2. Peptides
ues of TLPs
for
(b) En were
queluted
posiciones
podran
enlaces
with a linear se
gradient
from 0 toformar
60% acetonitrile
rate concentrain 30 min at a flow rate of 1 ml!min. Absorption of the eluting
values (21).disulfuro
The
intracatenarios?
peptides was monitored at 254 nm.
ned by incubatBacillus licheniformis !-amylase (1 mg!ml) in 50 mM Mops,
s!HCl, pH (c)
7.5!5
pH 7.0!5 mM CaCl !0.01% Triton X-100 was incubated with

min, after which

determined as

purified TLP-ste (1 "g!ml), the 8-fold mutant, or without

protease for 60 min at different temperatures. The reaction
volume was 500 "l. After incubation the samples were cooled
on ice, which resulted in aggregation of the substrate in the
samples that had been incubated at 100C. Precipitates (only
observed in the 100C samples) were collected by centrifugation and redissolved in 500 "l 6 M urea. Both supernatants and
redissolved precipitates were subjected to standard SDS!
PAGE, including pretreatment with sample loading buffer (5
min at 100C). The samples were identical in size (20 "l
supernatant and 20 "l dissolved precipitate). Gels were stained
with Coomassie brilliant blue.

nation of their
mM NaAc, pH
ropanol!0.01%
n at the various
and transferred
ual activity was
rate in 50 mM
previously (11).
ng agents were
CaCl2!50 mM
Indique la posible localizacin, ya sea en el
using 100 "M
e amide) as ainterior de la protena o en su superficie externa,
RESULTS AND DISCUSSION
gents were preThesiguientes
amino acid substitutions
A4T, T56A, G58A,
min before thede los
aminocidos:
Asp,T63F,
Ile,S65P,
Thr, Ala,
llowed by meaA69P, G8C, and N60C were combined in TLP-ste, and the

Gln, Lys. Explique su razonamiento.

(d) Qu aminocidos estn en los
extremos C- y Nmatic properties at 37C
Stability
terminal
de
la
protena?
K
T
Half-lifes
k !K

d-type TLP-ste, thermolysin, and the 8-fold mutated variant

cat

!3

opt

FaAFa
(M!1!s!1) #10!3

Phosphoramidon,
nM

Casein,
C

80C
(min.)

222 $ 33
NA
266 $ 23

50 $ 18
NA
43 $ 13

74
77
95

17.5
&200
Stable

90C
(min.)
1.5
12.5
Stable

100C
(min.)
%0.5
1
170