Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids

Eftink, Maurice R.; Ghiron, Camillo A.; Kautz, Roger A.; Fox, Robert O.

doi:10.1021/bi00219a005

Cited by 47 publications

(55 citation statements)

References 26 publications

(29 reference statements)

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“…A comparison of these values with our values is summarized in Table 8. Eftink et al (1991) observed the unfolding of H124L at pH 7.2 using fluorimetry and obtained a value for AH,, smaller than ours by roughly the same factor as listed for V66L at the higher temperatures in Table 7.…”

Section: Discussionsupporting

confidence: 65%

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry

1993

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The effects of eight mutations on the thermodynamics of the reversible thermal unfolding of staphylococcal nuclease have been determined over a range of pH and protein concentration by means of differential scanning calorimetry. Variation of the protein concentration was included in our study because we found a significant dependence of the thermodynamics of protein unfolding on concentration. Values for the change in the standard free energy of unfolding, AAG:, produced by the mutations in the pH range 5.0-7.0 varied from 1.9 kcal mol" (apparent stabilization) for H124L to -2.8 kcal mol" (apparent destabilization) for L25A. As has been observed in numerous other cases, there is no correlation in magnitude or sign between AAG: and the corresponding values for AM,, and TAAS:, the latter quantities being in most cases much larger in magnitude than AAG:. This fact emphasizes the difficulty in attempting to correlate the thermodynamic changes with structural changes observed by X-ray crystallography.Keywords: differential scanning calorimetry; mutations; staphylococcal nuclease; thermal unfoldingThe thermal denaturation of staphylococcal nuclease (SNase) and several of its mutant forms has been studied by Shortle et al. (1988) using fluorescence emission to monitor the thermally induced conformational changes. The data were subjected to van't Hoff analysis to obtain the thermodynamic parameters for the denaturations. The denaturations were observed over a range of pH, which led to a variation in the mid-temperature of denaturation of approximately 30 "C and permitted evaluation of the permanent change in heat capacity accompanying the denaturations.We have undertaken a reinvestigation of the thermal unfolding of five of these mutants-V66L, G79S, G88V, V66L + G88V, and V66L + G79S + G88V-using differential scanning calorimetry (DSC). We have also included in our study H124L, a naturally occurring form obtained from the V8 strain of Staphylococcus aureus, and the mutants L25A and A90S. It was also necessary to do a careful study of the wild-type (WT) enzyme for comparison

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Section: Discussionsupporting

confidence: 65%

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry

1993

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“…Biochemical Evidence for an H1/H4 Hydrophobic InterfaceThe predicted EF-hand pair in the Na V 1.5 COOH terminus contains a single tryptophan, Trp 1798 , which is important not only because our model predicts it to be integral to the hydrophobic interface but also because its intrinsic fluorescence can be used to probe and report the nature of the environment in which it is located (13,24,25). We thus prepared a GST fusion protein consisting of residues predicted to form the EF-hand pair (residues 1786 -1863) and carried out experiments using Trp 1798 as a reporter of its environment.…”

Section: Thermodynamic Cycle Analysis Is Consistent With Predictedmentioning

confidence: 99%

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Glaaser

Bankston

Liu³

et al. 2006

Journal of Biological Chemistry

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Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart Na V 1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the Na V 1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na ؉ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it.Channelopathies, so named because they represent a set of diseases caused by mutations in genes coding for ion channels, are a new and growing class of human disorders that include but are not limited to diabetes, muscle disorders, neurological disease, and cardiac arrhythmias (1). A surprising number of channelopathies associated with a wide diversity of human disease are caused by similar mutation-induced changes in ion channel function. Inactivation of voltage-dependent Na ϩ channels is an example of a physiological process critically important in many tissues that, when altered by mutation, can result in muscle weakness, inherited epilepsies, autism, or cardiac arrhythmia (2-4). Clinical consequences of inherited mutations that disrupt Na ϩ channel inactivation provide the most direct link between ion channel biophysics and human physiology and pathophysiology. Conversely, investigation into the consequences of inherited mutations on ion channel function has, in many cases, provided insight into the physiological importance of novel regions and/or structures of ion channel proteins.In the heart, Na ϩ channels (Na V 1.5) 4 primarily underlie action potential initiation and propagation but more recently have been shown to be critical determinants of action potential duration, particularly in the setting of certain inherited channelopathies. Inherited mutations in SCN5A, the gene coding for Na V 1.5, are now known to underlie multiple inherited cardiac arrhythmias, including the congenital long QT syndrome variant 3, Brugada syndrome, and isolated conduction disease (5), and in most cases, these inherited mutations disrupt channel inactivation.Fast inactivation of Na ϩ channels is due to rapid block of the inner mouth of the channel pore by the cytoplasmic linker between domains III and IV that occurs within milliseconds of membrane depolarization...

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“…Our observation of significant destabilization by loop replacements is not unprecedented, however. Two Staphylococcus nuclease mutants constructed by replacing a fiveresidue 0-turn with a @-turn sequence from concanavalin A were found to be destabilized by about 4.5 kcal/mol with a corresponding reduction in T, of about 20 "C (Eftink et al, 1991). A mutant constructed by replacing the loop region of chymotrypsin inhibitor 2 with a nonapeptide sequence found in an a-helix in subtilisin Carlsberg is also significantly destabilized by about 8 kcal/mol (Osmark et al, 1993).…”

Section: Loop Swaps and Destabilizationmentioning

confidence: 99%

Destabilizing loop swaps in the CDRs of an immunoglobulin V_L domain

Helms

Wetzel

1995

Protein Science

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It is generally believed that loop regions in globular proteins, and particularly hypervariable loops in immunoglobulins, can accommodate a wide variety of sequence changes without jeopardizing protein structure or stability. We show here, however, that novel sequences introduced within complementarity determining regions (CDRs) 1 and 3 of the immunoglobulin variable domain REI VL can significantly diminish the stability of the native state of this protein. Besides their implications for the general role of loops in the stability of globular proteins, these results suggest previously unrecognized stability constraints on the variability of CDRs that may impact efforts to engineer new and improved activities into antibodies.Keywords: complementarity determining regions; hypervariable loops; immunoglobulin stability; protein stability; Stern-Volmer plots; unfolding In the structures of antibody heavy and light chain variable domains, complementarity determining region (CDR) loops of diverse sequence and conformation project from P-sandwich frameworks to determine the affinity and specificity of antigen binding. These diverse sequences arise in the development of an antibody response via a complex combination of gene rearrangements and somatic mutations (Branden & Tooze, 1991). Protein engineers have transferred antigen binding activity from one framework to another by swapping hypervariable CDR loops between frameworks (Jones et al., 1986;Riechmann et al., 1988). In addition, totally artificial loop sequences have been introduced into variable domain frameworks to generate novel binding functions (Barbas et al., 1993;Fisch et al., 1994). This apparent tolerance of the immunoglobulin fold for both sequence diversity and loop swaps is consistent with the view that the major determinants of structural stability of globular proteins lie in the sequence-specific formation and packing of regular secondary structure elements (Shortle, 1992;Matthews, 1993), whereas surface loops are more forgiving of sequence changes (EL Hawrani et al., 1994).Although the CDR loops differ greatly in sequence, they are not infinitely variable in either sequence or conformation. Positions with highly conserved amino acids are found within many CDR sequences (Kabat et al., 1991 of "canonical structures" appear to be available for each CDR. For example, in an examination of the structural database available in 1989, only four different loop conformations were identified in the light chain crystallographic database for CDRl, and only three for CDR3 . If these patterns d o suggest constraints on the sequence variability of CDRs, however, it remains difficult from examination of evolved antibodies to either gauge the severity of these constraints or to understand their structural or functional basis.In this report we describe the preparation of a number of mutants of the immunoglobulin light chain variable domain REI, in which wild-type CDR sequences were replaced by loops of differing sequence and length. These replacements cause dramat...

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Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids

Cited by 47 publications

References 26 publications

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Destabilizing loop swaps in the CDRs of an immunoglobulin V_L domain

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Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids

Cited by 47 publications

References 26 publications

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry

Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Destabilizing loop swaps in the CDRs of an immunoglobulin VL domain

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Destabilizing loop swaps in the CDRs of an immunoglobulin V_L domain