Core mutants of the immunoglobulin binding domain of streptococcal protein G: Stability and structural integrity

Gronenborn, Angela M.; Frank, MM; Clore, G. Marius

doi:10.1016/s0014-5793(96)01262-8

Cited by 30 publications

(36 citation statements)

References 31 publications

(34 reference statements)

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“…Recently, we used a thermodynamic cycle comprising k obs measurements of residues in chymotrypsin inhibitor 2 and a variant, in buffer and in reconstituted cytosol, to confirm the EX2 mechanism (26). We repeated this strategy with the destabilized GB1 variant, I6L (57 (Fig. 7 and Fig.…”

Section: Resultsmentioning

confidence: 99%

Residue level quantification of protein stability in living cells

Monteith¹,

Pielak

2014

Proc. Natl. Acad. Sci. U.S.A.

108

178

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Significance Proteins function in a sea of macromolecules within cells, but are traditionally studied under ideal conditions in vitro . The more details we amass from experiments performed in cells, the closer we will get to understanding fundamental aspects of protein chemistry in the cellular environment. In addition to furthering our essential knowledge of biochemistry, advancements in the field of macromolecular crowding will drive efforts to stabilize protein-based therapeutics. Here, we show that protein stability can be measured at the residue level in living cells without adding destabilizing cosolutes or heat.

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Section: Resultsmentioning

confidence: 99%

Residue level quantification of protein stability in living cells

Monteith¹,

Pielak

2014

Proc. Natl. Acad. Sci. U.S.A.

108

178

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“…The S14 mutant of protein GB1 was obtained from a library of random core mutations 27,28 and comprises three changes, L5M, L7V and V54I. 13 C, 15 N-, 15 N-and non-labeled S14 proteins were over-expressed in Escherichia coli and purified as described.…”

Section: Methodsmentioning

confidence: 99%

Insights into Conformation and Dynamics of Protein GB1 During Folding and Unfolding by NMR

Ding

Louis

Gronenborn

2004

Journal of Molecular Biology

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“…The folding of the B1 domain has been studied extensively~Alexander et al, 1992b;Kuszewski et al, 1994;Orban et al, 1994Orban et al, , 1995Kobayashi et al, 1995;Park et al, 1997;Blanco & Serrano, 1998;Sheinerman & Brooks, 1998! and the B1 domain has been used as a model system for the selection and design of modified hydrophobic core structures~O 'Neil et al, 1995;Gronenborn et al, 1996;Dahiyat & Mayo, 1998!, to test the secondary structure propensities of the amino acids~Minor & Kim, 1994aKim, , 1994bSmith et al, 1994;!, to probe the influence of tertiary structural environment on secondary structure formation~Mi-nor & Kim, 1996!, and to evaluate the energetics of side-chainside-chain interactions~Smith & Regan, 1995!.…”

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confidence: 99%

The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G

et al. 2000

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The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15 N NMR-derived order parameters. Longitudinal~R 1 ! and transverse~R 2 ! relaxation rates, transverse cross-relaxation rates~h xy !, and steady state $ 1 H%-15 N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 8C for 89-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R 2 0h xy was used to identify nuclei for which conformational exchange makes a significant contribution to R 2 . Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter~S 2 ! was used to calculate the contribution of each NH group to conformational heat capacity~C p ! and a characteristic temperature~T *!, representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ;0.8 kJ0mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ;9 8C lower~78 8C rather than 87 8C!. Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.Keywords: B1 domain; entropy; heat capacity; NMR relaxation; order parameter; protein dynamics; protein stability Most globular proteins are marginally stable because the factors that favor formation of the native state, primarily desolvation of hydrophobic groups and formation of intramolecular hydrogen bonds and salt bridges, are almost equally balanced against those that favor denaturation, primarily the higher conformational entropy of the unfolded protein chain relative to that of the native state~Creigh-ton, 1993; Fersht, 1999!. At high temperatures, the balance between these factors is altered such that many proteins exhibit reversible thermal denaturation with a characteristic midpoint, the melting temperature~T m !. The free energy of unfolding of a proteiñ DG NϪU ! depends upon the changes in enthalpy, entropy, and heat capacity that occur upon unfolding and the temperature~T ! according to the equation~Creighton, 1993; Fersht, 1999!:in which DH 0 and DS 0 are the enthalpy and entropy changes, respectively, at a reference temperature T 0 and DC p,NϪU is the change in heat capacity at constant pressure, which is assumed to be invariant with temperature. Equation 1 indicates that native proteins may be stabilized by an increase in DH 0 or by a decrease in either DS 0 or DC p,NϪU . Thus, one possible strategy for a protein to achieve high thermal ...

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Core mutants of the immunoglobulin binding domain of streptococcal protein G: Stability and structural integrity

Cited by 30 publications

References 31 publications

Residue level quantification of protein stability in living cells

Residue level quantification of protein stability in living cells

Insights into Conformation and Dynamics of Protein GB1 During Folding and Unfolding by NMR

The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G

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