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Thermodynamics of protein denaturation at temperatures over 100?°C: CutA1 mutant proteins substituted with hydrophobic and charged residues.

ABSTRACT: Although the thermodynamics of protein denaturation at temperatures over 100?°C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113?°C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td?=?137?°C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (?H) with no entropic gain from hydrophobic solvation around 100?°C, and that the stabilization due to salt bridges resulted from both the increase in ?H from ion-ion interactions and the entropic effect of the electrostatic solvation over 113?°C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.

SUBMITTER: Matsuura Y

PROVIDER: S-EPMC4620440 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Thermodynamics of protein denaturation at temperatures over 100 °C: CutA1 mutant proteins substituted with hydrophobic and charged residues.

Matsuura Yoshinori Y Takehira Michiyo M Joti Yasumasa Y Ogasahara Kyoko K Tanaka Tomoyuki T Ono Naoko N Kunishima Naoki N Yutani Katsuhide K

Scientific reports 20151026

Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mut ...[more]

PMID: 26497062

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Project description:BACKGROUND: The three-dimensional structure of a protein can be described as a graph where nodes represent residues and the strength of non-covalent interactions between them are edges. These protein contact networks can be separated into long and short-range interactions networks depending on the positions of amino acids in primary structure. Long-range interactions play a distinct role in determining the tertiary structure of a protein while short-range interactions could largely contribute to the secondary structure formations. In addition, physico chemical properties and the linear arrangement of amino acids of the primary structure of a protein determines its three dimensional structure. Here, we present an extensive analysis of protein contact subnetworks based on the London van der Waals interactions of amino acids at different length scales. We further subdivided those networks in hydrophobic, hydrophilic and charged residues networks and have tried to correlate their influence in the overall topology and organization of a protein. RESULTS: The largest connected component (LCC) of long (LRN)-, short (SRN)- and all-range (ARN) networks within proteins exhibit a transition behaviour when plotted against different interaction strengths of edges among amino acid nodes. While short-range networks having chain like structures exhibit highly cooperative transition; long- and all-range networks, which are more similar to each other, have non-chain like structures and show less cooperativity. Further, the hydrophobic residues subnetworks in long- and all-range networks have similar transition behaviours with all residues all-range networks, but the hydrophilic and charged residues networks don't. While the nature of transitions of LCC's sizes is same in SRNs for thermophiles and mesophiles, there exists a clear difference in LRNs. The presence of larger size of interconnected long-range interactions in thermophiles than mesophiles, even at higher interaction strength between amino acids, give extra stability to the tertiary structure of the thermophiles. All the subnetworks at different length scales (ARNs, LRNs and SRNs) show assortativity mixing property of their participating amino acids. While there exists a significant higher percentage of hydrophobic subclusters over others in ARNs and LRNs; we do not find the assortative mixing behaviour of any the subclusters in SRNs. The clustering coefficient of hydrophobic subclusters in long-range network is the highest among types of subnetworks. There exist highly cliquish hydrophobic nodes followed by charged nodes in LRNs and ARNs; on the other hand, we observe the highest dominance of charged residues cliques in short-range networks. Studies on the perimeter of the cliques also show higher occurrences of hydrophobic and charged residues' cliques. CONCLUSIONS: The simple framework of protein contact networks and their subnetworks based on London van der Waals force is able to capture several known properties of protein structure as well as can unravel several new features. The thermophiles do not only have the higher number of long-range interactions; they also have larger cluster of connected residues at higher interaction strengths among amino acids, than their mesophilic counterparts. It can reestablish the significant role of long-range hydrophobic clusters in protein folding and stabilization; at the same time, it shed light on the higher communication ability of hydrophobic subnetworks over the others. The results give an indication of the controlling role of hydrophobic subclusters in determining protein's folding rate. The occurrences of higher perimeters of hydrophobic and charged cliques imply the role of charged residues as well as hydrophobic residues in stabilizing the distant part of primary structure of a protein through London van der Waals interaction.

Project description:UNLABELLED:The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a ?-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of ?-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the ?-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general. IMPORTANCE:HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr?Phe or Asp?Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

Dataset Information

Thermodynamics of protein denaturation at temperatures over 100?°C: CutA1 mutant proteins substituted with hydrophobic and charged residues.

Publications

Thermodynamics of protein denaturation at temperatures over 100 °C: CutA1 mutant proteins substituted with hydrophobic and charged residues.

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