Chapter 5

Physical Interactions That

determine that Properties

of Proteins

A. Enzyme deactivation by chemical process:

oxidation, heat inactivation, pH, metal ion,ionic

strength, protease

B. Enzyme Inactivation caused by environmental

conditions:

- adsorption of inhibitors

- bacteria contamination

- leakage of enzyme from support : especially bad

for medicinal purposes

- decomposition of support : use strong supports

when possible

5-1 Decay of Biocatalyst Activity

Reversible vs. Irreversible Inactivation

A. reversible inactivation: enzyme activity returns to original level

B. irreversible inactivation:

이와 같은 비가역적 실활을 방지하기 위해서는 해당 아미노산(예: Asp,

Asn, Cys, Gln등)을 다른 아미노산으로 치환하는 방법이 한가지이지만

gene cloning, site-directed mutagenesis등이 선결되어야 하므로 매우

오랜 시간과 노력이 필요하다.

가역적 실활의 경우는 가해진 처리방법을 바꾸어주거나 조정해주면 쉽게

효소의 활성이 되돌아오기 때문에 간단하게 문제를 극복해 줄 수 있으며 일

반적으로는 효소의 고정화 (흡착, 공유결합을 이용)를 시도하며 효소의 가교

결합, buffer종류 및 strength변화를 통한 pH의 변화, 단백질 안정제의 사

용(예: 염, chelating agent등), 적절한 온도의 사용 등을 생각할 수 있다.

<효소의 비가역적 실활>

5-2 A. 안정화의 중요 결정 인자들 :

proteins fold spontaneously at const. T & PA. 접힘 공정의 주요 인자들

소수성(hydrophobic force) > disulfide bond > 수소결합(hydrogen bond)

B. 구조적 관점에서 주요 인자들

보통 단백질은 일정 온도와 압력에서 자발적으로 접힘(folding)이 진행되어 안정한모양을 갖게되는데 이때 안정화되는 에너지가 약 ΔG=10-20~kcal/mol 정도 된다고한다. 이에 관여하는 아미노산 분자들간의 힘은 다음과 같은 결합 내지는 힘에 의해결정된다.

수소 결합(hydrogen bond)

Disulfide bond

정전기적 결합(electrostatic bond such as ion pair, dipole b/w

amide and helix, dipole b/w helixes, forces b/w helix and ion,

etc.)

소수성 결합(hydrophobic bond)

구조적 엔트로피 변화(conformational entropy change)

무극성기의 상호 작용(non-polar interaction)

기타(repulsive force, dispersion force)

(a) H-bonding

Oxygen atoms participate simultaneously as acceptors in two H bonds.

N-H –C(=O) - - -H-N

2ndary structures: alpha helix and beta-sheet

40-70% of main chain C=O and N-H group

H-bondings formed by external side chains are weak

6kcal/mol of H-bonding in vacuum , 2-10kcal/mol at RT in water

( in reality, > 3kcal/mol, side chain H-bonding exerts ca. 0.5-1.5 kcal/mol)

protein folding 시 H-bonding에 의한 free energy의 변화는 매우 적다:

Because H-bondings between water and H-bonding donors/acceptors are

replaced by H-bonding b/w proteins

Therefore, H-bonding is important for forming 2ndary, tertiary structure of

protein, but not for overall stability.

However, If there are many H-bondings as such, their sums become

important. e.g.) conformational entropy

5-2 B. 안정화의 중요 결정 인자들 :

5-2 C. 안정화의 중요 결정 인자들 :

(b) S-S bonds

SH-P-SH + RSSR P-S-S + 2RSH

Intra-chain disulfide bond is formed while the protein is still folding

When the two bridges are formed, it goes through complex disulfide

rearrangements

The energy of a S-S bridge relative to two thiol groups depends upon

the redox-potential of the environment

Disulfide bridges themselves can stabilize a folded protein

conformation.

Interaction b/w Dipoles Summary

Interactions between dipoles(m1=q1*l)and charge q2

(c) Electrostatic forces

1. Ion pairs: 1/ εr

- Two Point charges: two charges separated by a distance (r) in a

medium of dielectric constant (ε)

E=ZaZb q2/ εrab: Coulomb’s law

e.g.) H2O= 80, solvent= 2-110

38% of Arg, 29% of His, 16% of Asp, and 20% of Lys

most pairs are distant in the sequence, and in different segments of

secondary structure

about 85% of ion pairs are at the protein surface

do not exert a crucial influence on the stability of structures or on a

pathway of folding

5-2 D. 안정화의 중요 결정 인자들 :

2. Dipoles: Energy of a charge q interacting with a dipole moment mD

which is separated by r and forms an angle(θ) with the line from the

dipole to the dipole to the charge

E=q.mD cos θ/ (ε*r2), (from negative to positive charge)

e.g.) Definition of unit : one e unit of positive and negative charge

separated by 1Å = 4.8 Debye units(D)

- Parallel dipoles repel each other

- Antiparallel dipoles attract each other

[cf.] Amide and helix dipole

C(=O)-NH represents 3.5 D=1.2*10-29Coulomb-meter, ref) H2O=1.85D

about 2X of dipole moment of water

alpha-helix: 0.5-0.75 Dunits + charge at N-terminus, - charge at C-

terminus

parallel beta sheet also has an effective dipole, but very small

3. Interactions of helix dipoles: 1/ (εr3), b/w two stationary dipoles

alpha-helix aligned in antiparallel fashion

antiparallel beta sheets and beta sheets and alpha helix

4. Interactions of helix dipoles with ions: 1/ (εr2)

position - ions towards N-termini of alpha-helix

e.g.) phosphate in low M.W. ligands bound to proteins at or near the

N-terminus of alpha-helix

Asp, Glu are more frequently found toward N-terminus of alpha-helix

(d) Hydrophobic interaction

Ile, Leu, Val, Trp, Tyr, and Phe

tendancy of nonpolar cmpds such as hydrocarbons to transfer from an

aqueous soln to an organic phase

measurement of hydrophobicity : partition of a molecule between

organic(n-octanol) and aqueous(H2O) phases

1. Hansch Eq’n ---hydrophobicity of small groups

hydrophovicity const for R(= p)

p = log P/P0, G= -RT ln(P0/P)=2.303 RT* p

P = ratio of the solubility of parent compounds(H-S) in the organic

phase octanol to that in aqueous phase

P0 = ratio of the solubility of compounds (R-S) in the organic phase

octanol to that in aqueous phase

i) additive effects are given

ii) nitro and amino groups conjugated with benzene ring depends

upon the other groups attached to the ring.

5-2 D. 안정화의 중요 결정 인자들

2. Hydrophobicity varies as surface area

empirical correlation b/w surface area of a hydrophobic side chain of A.A.

and G of transfer from water to an organic phase

e.g.) 1A2 of surface area gives G=80-100J/mol (20-25kcal/mol)

Calculation: surface tension of water = 72 dyne/cm(= 0.072 N/m),

E= force * distance, to form an area in water(= 1 Å * 1 Å *72 dyne/cm)

(to form a free 1 A2 of surface of water requires 7.2*10-22 J

(=1.72*10-22cal)), when this value is multiplied by Avogadro’s number,

E= 435 J/(A2/mol)= 104 cal/(A2/mol)~ energy which can create cavity

in water to be occupied by a hydrophobic group.

thermodynamics of transfer of a nonpolar solute(e.g. hydrocarbon) from a

nonpolar solvent to water, i.e. a polar solvent

endergonic(transferG > 0) process , exothermic (transferH < 0),

therefore, large negative in transferS <0

G = H –TS , H(-) < -TS(+)

3. hydrophobic interaction decreases in magnitude at lower

temperature.(ref. Fig. 4.9)

Cp= H/T = TS/T is proportional to nonpolar surface area of

solute

Unfavorable entropy changes result from increased ordering of water

molecules around nonpolar molecules

Cp increases, as Temp increases : As the temp increased, the

ordered water shell around the nonpolar solute tends to melt out and

to become more like bulk water, which causes large Cp of aqueous

solutions of nonpolar molecules

Cp is proportional to the non-polar surface area of the solute molecule

exposed to water

at Ts=140C (Str =0)at TH= 20C, (Htr =0)

The magnitude of hydrophobic interaction(Gtr)

= Htr - T Str

the difference in the magnitude of non-

covalent interactions b/w molecules that occur in

the two phase = Htr

@ If Gtr is >0 , the non-

polar molecules prefer a

non aqueous solution.

Thermodynamics of Hydrophobic Interaction

Ref: G =H-TS

G is maximum at S =0

TsTH

As accessible surface area

increases, : G(= hydrophobic

interaction) increases

(e) Other forces

1. Valance forces that result in the covalent bonds that hold the polypeptide

together.

2. Dispersion( or London) forces: weak, attractive force between

noncovalently bonded atoms, which is between two mutually induced dipoles.

= f(1/r6)

3. Repulsive forces by all noncovalently bonded atoms. When their electron

clouds overlap as they are brought within the sum of Van der Waals radii. =

f(1/r12)

Van der Waals radius= minimal estimate of the size of a molecule

Ref) P = RT/(Vm-b) – a/Vm2, b= volume taken up by the molecules

Van der Waals surface area and volume explain accessible surf. Area

(r H2O=1.4 Å ) Combining 2. and 3. is called Van der Waals interaction

ref) Lennard-Jones potential : E(potential energy)=A/r12 – B/r6,

optimum distance for the interaction of two atoms, given by the minimum, is

0.3-0.5 Å greater than the sum of Van der Waals radius.

2) 안정화의 중요 결정 인자들 :

(f) Conformational Entropy

Entropy change for unfolding: ΔSc=kB*ln[U/N]

kB= Boltzmann’s const

N= number of arrangements of the folded protein

U= number of arrangements in the same protein with unfolded state

The conformational entropy is usually positive, which relate

conformational entropy to stabilizing terms in the free energy.

G = H - TS, As T increases, TS term contributes more

e.g.) overall standard free energy of folding Gnet

Gnet = Gu( unfold form) + {G1 + G2 + …}

usually Gu > 0, Gn(n- interaction) < 0, | Gn|>|Gu|

therefore, protein is stable only if Gnet<0

2) 안정화의 중요 결정 인자들 :

C. 실활 관점에서 주요 인자들 온도(Temp)

산가(pH)

이온 세기(Ionic strength)

안정제 및 변성제(stabilizer and denaturant)

산소(Oxygen)

단백질 분해 효소(protease)

금속이온(Metal ion)

전단 응력(shear force)

2) 안정화의 중요 결정 인자들 :

3) 거시적 관점에서의 단백질 안정화

다시 말하면 이와 같은 힘들이 단백질의 안정성 결정에 중요한 역할

한다는 것을 알 수 있다. 간단하게 단백질의 접힘과 풀림(unfolding)을

이상계(two-state)모델로 나타내면

N U

N: nature

U: unfolded species

y: 측정값(예: O.D at 287nm )

f : fraction

접힘과 풀림 상태의 평형상수

K = fN / (1-fN ) = fN / fU

로 나타낼 수 있으며 접힘에 필요한 안정화 에너지 ΔG0 = -RT lnK

로 표시할 수 있고 urea나 GuHCl 용액 혹은 온도 변화 등을 통해서

실험적으로 구할 수 있다.

단백질이 완전히 실활하기 전에 풀리는 상태의 존재를 감안하는 경우는

K

N N' D

via i) conformational aggregation, ii) cleavage hydrolysis)

N:natural, N': unfolded, D: denatured

단백질의 변성 속도는 아래와 같이 유도되며 ,

V= -d[N]/ dt = d[D]/dt =k [ N’]

K= N’/ N , C0 = N+N'+D = Constant 이므로,

변성 단백질은 D=C0 ( 1- e (kK /1+K) t ) = C0(1-e kappt), kapp = (k*K /1+K)