Structural determinants of cold adaptation and stability in a large protein.


Abstract

The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable alpha-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T(m), the calorimetric enthalpy Delta H(cal), the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k(cat) and K(m). The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both k(cat) and K(m) by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted alpha-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation. Study holds ProTherm entries: 11440, 11441, 11442, 11443, 11444, 11445, 11446, 11447, 11448, 11449, 11450, 11451, 11452, 11453, 11454, 11455, 11456, 11458, 11459, 11460, 11461 Extra Details: two-state transition; weak interactions; cooperativity;,thermal inactivation rate constant; catalysis

Submission Details

ID: reuxPkiE3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:42 p.m.

Version: 1

Publication Details
D'Amico S;Gerday C;Feller G,J. Biol. Chem. (2001) Structural determinants of cold adaptation and stability in a large protein. PMID:11325973
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AQH 1997-07-30T00:00:00+0000 2.0 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS
1AQM 1997-07-31T00:00:00+0000 1.85 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS COMPLEXED WITH TRIS
1B0I 1998-11-10T00:00:00+0000 2.4 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS
1G94 2000-11-22T00:00:00+0000 1.74 CRYSTAL STRUCTURE ANALYSIS OF THE TERNARY COMPLEX BETWEEN PSYCHROPHILIC ALPHA AMYLASE FROM PSEUDOALTEROMONAS HALOPLANCTIS IN COMPLEX WITH A HEPTA-SACCHARIDE AND A TRIS MOLECULE
1G9H 2000-11-23T00:00:00+0000 1.8 TERNARY COMPLEX BETWEEN PSYCHROPHILIC ALPHA-AMYLASE, COMII (PSEUDO TRI-SACCHARIDE FROM BAYER) AND TRIS (2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL)
1JD7 2001-06-13T00:00:00+0000 2.25 CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300R OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE
1JD9 2001-06-13T00:00:00+0000 2.5 CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300Q OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE
1KXH 2002-01-31T00:00:00+0000 2.3 Crystal structure of the complex between an inactive mutant of psychrophilic alpha-amylase (D174N) and acarbose
1L0P 2002-02-12T00:00:00+0000 2.1 CRYSTAL STRUCTURE ANALYSIS OF THE COMPLEX BETWEEN PSYCHROPHILIC ALPHA AMYLASE FROM PSEUDOALTEROMONAS HALOPLANCTIS AND NITRATE
1BVN 1998-09-16T00:00:00+0000 2.5 PIG PANCREATIC ALPHA-AMYLASE IN COMPLEX WITH THE PROTEINACEOUS INHIBITOR TENDAMISTAT

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Alpha-amylase P29957 AMY_PSEHA
98.4 Pancreatic alpha-amylase P00690 AMYP_PIG