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Published online before print April 5, 2006, 10.1110/ps.052018006
Protein Science (2006), 15:1085-1092. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Sequential reorganization of beta-sheet topology by insertion of a single strand

Martin Sagermann1, Walter A. Baase and Brian W. Matthews

Institute of Molecular Biology, Howard Hughes Medical Institute, and Department of Physics, University of Oregon, Eugene, Oregon 97403-1229, USA

(RECEIVED December 7, 2005; FINAL REVISION February 3, 2006; ACCEPTED February 3, 2006)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
Insertions, duplications, and deletions of sequence segments are thought to be major evolutionary mechanisms that increase the structural and functional diversity of proteins. Alternative splicing, for example, is an intracellular editing mechanism that is thought to generate isoforms for 30%–50% of all human genes. Whereas the inserted sequences usually display only minor structural rearrangements at the insertion site, recent observations indicate that they may also cause more dramatic structural displacements of adjacent structures. In the present study we test how artificially inserted sequences change the structure of the beta-sheet region in T4 lysozyme. Copies of two different beta-strands were inserted into two different loops of the beta-sheet, and the structures were determined. Not surprisingly, one insert "loops out" at its insertion site and forms a new small beta-hairpin structure. Unexpectedly, however, the second insertion leads to displacement of adjacent strands and a sequential reorganization of the beta-sheet topology. Even though the insertions were performed at two different sites, looping out occurred at the C-terminal end of the same beta-strand. Reasons as to why a non-native sequence would be recruited to replace that which occurs in the native protein are discussed. Our results illustrate how sequence insertions can facilitate protein evolution through both local and nonlocal changes in structure.

Keywords: sequence duplication; beta-sheet; T4 lysozyme; protein evolution


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Most proteins fold into a unique, sequence-specific architecture. Understanding the resultant topology has remained a prime challenge in modern biochemistry.

A number of proteins have been subjected to extensive mutagenesis and folding studies to identify the role of loops in protein folding (for example, see Ladurner and Fersht 1997; Viguera and Serrano 1997; Wright et al. 2004). These studies reveal that amino acid substitutions, deletions, and insertions are generally well tolerated in the loops of the proteins studied. Even though the length of an inserted loop sequence may have an effect on the stability and speed of folding, it generally does not have a significant effect on the overall structure of a protein. Circular permutation studies and the assembly of proteins by fragment complementation (Goldenberg and Creighton 1983; Iwakura et al. 2000; Smith and Matthews 2001) further demonstrate the passive role of loops in defining topology. In contrast to these studies, however, insertions and deletions within secondary structures are likely to have a dramatic effect on a structure. Such mutations can cause packing disruptions and register shifts of adjacent sequences (Heinz et al. 1993; Vetter et al. 1996).

Even though insertions appear to be well tolerated within loops of proteins, the insertion of entire secondary structures into loops can also have major effects on the overall structure of a protein. For example, the insertion of a beta-hairpin into a beta-sheet loop of the outer-surface-protein OspA (Koide et al. 2000) extends the single-layer sheet structure by two additional strands. In this case, the structure and topology of the adjacent structure, however, remained unchanged.

Recently, the insertion of a copy of an 11-residue helix of T4 lysozyme into a loop was shown to result in an alternatively folded structure in which the insert "looped out" and adopted a partially non-native conformation (Sagermann et al. 1999). By modifying the design, however, the insert could be recruited into the native architecture (Sagermann et al. 2003). In either case, the mutation had relatively little effect on the overall structure of the enzyme, suggesting that large insertions into loops could be tested at other sites.

In the present study we examine the consequences of insertions adjacent to beta-strands, rather than next to an {alpha}-helix. Specifically, we have targeted two different beta-strands in T4 lysozyme (Fig. 1). In both cases, a segment of the native sequence was copied and inserted into a loop structure adjacent to the parent sequence, thus creating an exact tandem repeat. In such a case, the close proximity of the two identical sequences might lead to alternative folds, as illustrated in Figure 2. For example, the structure of the parent sequence could remain the same and the inserted structure be forced to loop out (Fig. 2B). Other scenarios are also possible (e.g., Fig. 2C). Even in such a relatively simple situation, predicting the most likely outcome is not trivial.


Figure 1
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  		Figure 1. Schematic representation of the beta-sheet of T4 lysozyme. The sheet structure consists of Strands I, II, and III and turns T-1, T-2, and T-3. The sequences to be inserted are shown in red. In mutant L30c, the inserted amino acid sequence corresponds to Strand II plus Turn T-2 and is inserted after Tyr24. In mutant L31d, the inserted sequence corresponds to Turn T-2 plus Strand III and is inserted after residue Leu33. The color-coding shown here is maintained in all figures.

 


Figure 2
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  		Figure 2. Illustration of some possible folds of the beta-sheet domain as a result of the insertion L30c. The inserted sequence (red) contains Strand II and Turn T-2. (A) In the simplest scenario, the inserted structure loops out at the insertion site, i.e., at Turn T-1. The neighboring structure retains the native conformation, leaving the identical parent sequence (yellow) unchanged. (B) In a second scenario, the insert sequence displaces the identical parent sequence, forcing it to loop out at Turn T-2. (C) In yet another scenario (the one that is observed), the sequence that is displaced in B continues so as to displace the sequence in Strand III. The structure will then be forced to loop out at T-3. In going from scenario B to C, the wild-type turn structure T-2 (Gly-Ile-Gly) is restored. In contrast, however, Strand II now replaces Strand III, causing substitutions in this region.

 
Design strategy
The N-terminal domain of T4 lysozyme contains a somewhat irregular beta-sheet comprising Strands I, II, and III. These are connected, according to the definition of Kabsch and Sander (1983), by tight turns, T-1, T-2, and a loop T-3 (Fig. 1).

The two duplication mutants presented here were derived from Strands II and III and the turn that connects them (Fig. 1). Mutant L30c is the result of the duplication of residues Tyr25 to Gly30 bearing the sequence of Strand II and Turn T-2. These six amino acids were inserted after residue Tyr24. Mutant L31d contains a duplication of residues Gly28 to Leu33, corresponding to Turn T-2 plus Strand III. This sequence was inserted after residue Leu33. The two loops T-1 and T-3 are surface-exposed, and looping out is unlikely to cause any steric clashes with the remaining structure. Loop T-2, however, is largely buried and packs against the long interdomain helix C of the enzyme. A looping-out event at this site would be likely to severely jeopardize the integrity of the entire structure. For this reason, all mutant structures were designed such that the sequence Gly-Ile-Gly would always be available to maintain the packing constraints irrespective of the folding scenario (Fig. 2). Consistent with our previously used nomenclature (Sagermann et al. 1999), inserted amino acids retain the original residue number appended with an "i."


   Results
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 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Both mutant proteins remained enzymatically active and crystallized readily. Crystals of L31d grew rapidly overnight into thin needles. Crystals of mutant L30c usually grew in clusters of very thin plates that typically diffracted to <3 Å resolution. Due to the poor crystal quality and low completeness of collected data, several data sets were collected for this mutant but could not be averaged because of variations in the unit cell dimensions. Consequently, the structure was independently refined using several different data sets so that sites of looping out could be identified reliably.

Structure of L30c
The overall structure of L30c remains very similar to wild-type T4 lysozyme with no obvious change in the hinge-bending angle (Fig. 3A). In the crystal lattice the four molecules per asymmetric unit are arranged as two dimers that are packed in a "back-to-back" arrangement, which is frequently observed in mutant lysozyme crystals (Zhang et al. 1995). Inspection of the N-terminal domain of the molecules does not reveal any major changes in the position of the beta-strands relative to the rest of the protein.


Figure 3
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  		Figure 3. (A) Stereo pair showing the superposition of the backbone of mutant L30c (cyan) on wild-type lysozyme (light gray). The color-coding of thebeta-sheet structure in the mutant is the same as that in Figure 1C. No electron density could be identified between Gly28 and Ser36. This region is indicated by the red asterisks. (B) Stereo representation of the beta-sheet region of mutant L30c, molecule C. The 2Fo-Fc map was calculated to 3 Å resolution and contoured at 0.8{sigma} above the mean. The structure between residues Lys16 and Ile29 is shown. The inserted sequence (red) now occupies the position of the native Strand II, whereas Strand II has displaced Strand III. (C) Superposition of the refined backbone of molecule A of mutant L30c on the wild-type structure in the vicinity of the beta-sheet. The inserted sequence is highlighted in red and the Strand II sequence in yellow. The structure of Strand III is shown as a dashed line, as it could not clearly be identified in the electron density. (D) Superposition of the backbone of molecule C of mutant L30c on the wild-type structure. The conformation of the T-1 loop differs somewhat from molecule A, but the overall structure is essentially the same. Despite a different conformation of loop T-1, no additional positive density in the vicinity could be detected that would indicate another loop-out site. Panels A and B were generated with PyMOL (DeLano Scientific LLC), and panels C and D were generated with MOLSCRIPT (Merritt and Murphy 1994; Esnouf 1997).

 
Even though the insertion occurs in Turn T-1, it appears that the major change in the structure occurs in Turn T-3. As can be seen in Figure 3B, there is continuous electron density starting at Lys16 and continuing through Strands I, II, and III. This region includes the duplicated residues (Y25i-G30i) that replace the original sequence (Y25-G30). Residues Y25–G30 in turn replace residues within Strand III, as indicated in scenario C of Figure 2. At the C terminus of Strand III (i.e., at the T-3 loop), the electron density becomes weak and uninterpretable for residues Gly28–Ser36, indicating that this region has looped out in an essentially disordered fashion (Fig. 3C). The conformations of all four molecules in the asymmetric unit are essentially the same, although there is some variability in the geometry of the T-1 loop (Fig. 3C,D).

Structure of L31d
Mutant L31d also has four molecules in the asymmetric unit, again arranged in pairs of dimers. In this case, they are related by a translation of ~49 Å. The structure was solved by molecular replacement using the mutant lysozyme I3P (Dixon et al. 1992) as a search model. In comparison to wild-type T4 lysozyme, this mutant displays a dramatically increased hinge-bending angle (Fig. 4A). Inspection of the refined structure of L31d indicates that the orientation of the N-terminal domain of all four molecules has rotated by ~42° relative to the C-terminal domain. This large hinge-bending angle allows for the long helix C to be packed in the active site of an adjacent molecule in the crystal.


Figure 4
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  		Figure 4. (A) Comparison of the backbone structure of mutant L31d (magenta) and wild-type (light gray). The alignment is based on the superposition of residues 38–61 of wild type and the corresponding residues in the mutant. Color-coding of the beta-sheet region is the same as in Figure 1C. (B) Stereo representation of the beta-sheet region of mutant L31d. Shown is the 2Fo-Fc electron density between residues 16 and 37 of molecule A. The density was calculated to 3 Å resolution and is contoured to 1.0{sigma} above the mean. The new loop forms a hairpin structure (red) that extends away from the native beta-sheets. (C) Deletion (Fo-Fc) electron density of the beta-sheet structure of L31d, molecule B. The density was contoured at +2.2{sigma} at 3 Å resolution. Residues 33–38 were omitted from the calculation. Even though weaker for molecules B and C than for A and D, the electron density for the loop structure is visible. Upon model building and refinement, additional density in the immediate vicinity appeared, suggesting multiple conformations of this region. Due to the low occupancy, the corresponding loop residues of molecules B and C were omitted from the final model. (D) C{alpha} representation of the beta-sheet of mutant L31d, molecule A (dark gray), superimposed onto the wild-type structure (light gray). The mutant structure is drawn in dark gray, and Strands I and II are in yellow and in green, respectively. The newly inserted sequence is shown in red. The insert structure forms a beta-hairpin almost perpendicular to the original beta-sheet structure. The structure is stabilized through interactions with a symmetry-related molecule (not shown). Panels A, B, and C were generated with PyMOL, and panel D was generated with MOLSCRIPT/RASMOL.

 
For two of the molecules in the asymmetric unit (A and D) there is clear density showing the location of the duplicated residues (G28i–L33i) (Fig. 4B). They form a hairpin loop located at the site where the extra amino acids are inserted (Fig. 4D). This loop appears to be stabilized by an interaction between the side chain of L32i and a hydrophobic pocket formed by Asn2, Trp159, and Ala97 of a neighboring molecule in the crystal. Lys35, Ser36, and Pro37 adopt somewhat different non-native conformations to accommodate the new loop (Fig. 4D). His31 maintains a salt-bridge interaction with Asp70, as in the wild-type structure. The remaining structure of the N-terminal domain is unchanged. For molecules B and C, the electron density at the site of the insertion is weak (Fig. 4C), suggesting that the sequence duplicate is largely disordered.


   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
The simplest scenario for a protein to accommodate an insert is for the added amino acids to loop out at the insertion site, leaving the original interactions in the vicinity unchanged. This occurs for mutant L31d. All of the native residues required to maintain the original fold remain the same. The inserted sequence does not replace any existing loop or strand. Rather, it forms a beta-hairpin that is either disordered or stabilized in the crystal by interactions with a symmetry-related molecule (Fig. 4D).

In contrast, the accommodation of the duplicated sequence in mutant L30c is more complicated. In this case, the insert displaces Strand II and Turn T-2. In turn, this displaced sequence causes a further displacement of Strand III and forces Turn T-3 to loop out (Fig. 3C). This domino effect of strand displacements results in two consecutive strands in the beta-sheet having identical sequences (as in Fig. 2C). All four copies of the mutant in the crystals display a large change in hinge-bending angle (Fig. 4A), but this by itself is not unusual. Different mutants of T4 lysozyme display a wide range of hinge-bending angles that are presumably facilitated by different crystal contacts and reflect substantial conformational flexibility in solution (Zhang et al. 1995). Likewise, the large majority of these mutants retain catalytic activity, as is the case for L31d.

Stability results for L30c and L31d are largely consistent with this structural picture. The L30c insertion reduced the melting temperature 10°C for an estimated {Delta}{Delta}G of –3.7 kcal/mol. For L31d, the insertion reduced the melting temperature 8°C for an estimated {Delta}{Delta}G of ~–3 kcal/mol. In neither case was refolding facile under standard in vitro conditions, but it was more so for L30c at low concentration. In general terms, the loss of stability in both mutants is consistent with the disruption of wild-type interactions. For example, in neither case are the crystal structures consistent with a salt bridge from E22 to R137. This bridge has been found to contribute ~1 kcal/mol stabilization to the wild-type protein (DuBose et al. 1999). In mutant L30c the displacement of Strand III by the former Strand II replaces His31 by Tyr25, Leu32 by Thr26, and Leu33 by Ile27 (Figs. 1, 2). It is known that the substitution L32T causes only a small (0.3 kcal/mol) reduction in stability (DuBose et al. 1999). The replacement of Leu33 with the similar amino acid Ile would also be expected to be well tolerated. The replacement of His31 with a tyrosine, however, was not expected since it eliminates the salt bridge between Asp70 and His31. The measured stability of this salt bridge is 3–5 kcal/mol (Anderson et al. 1990), which is comparable with, if not greater than, the overall loss in stability of the mutant (3.7 kcal/mol). Possibly the tyrosine at site 31 replaces some of the interactions associated with the loss of the histidine. The substitutions associated with the strand insertion and displacement cause only minor changes in structure and solvent accessibility of the N-terminal domain despite the loss of the salt-bridge interaction.

Overall, the structures of the insertion mutants demonstrate that the wild-type scaffold of T4 lysozyme is maintained. In both mutants the looping out occurs in the T-3 loop and does not cause disruptions within the strands of the beta-sheet. The looping-out point is in both cases a glycine residue (Gly28 in L30c [Fig. 3C], Gly28i in L31d [Fig. 4D]). In molecules A and D of mutant L31d the looped-out residues Ile29i-Gly30i and Leu33i-Thr34 form two antiparallel hydrogen-bonded strands (Fig. 4D). This structure, however, is stabilized by favorable crystal contacts. The loops of molecules B and C as well as all four copies of mutant L30c appear to be largely disordered, suggesting that the looped-out structures do not fold into preferred autonomous conformations.

In previous experiments involving the duplication of an {alpha}-helix we observed that the competition to fold into the native structure appeared to involve only the parent sequence and its engineered tandem repeat (Sagermann et al. 1999, 2004). In contrast, the strand displacements observed in mutant L30c are not limited to the two identical sequence copies but also involve a neighboring strand that carries a different sequence. As a consequence, non-native amino acids are recruited to replace the native sequence, yet the wild-type fold is closely maintained. There are various possible reasons why this might occur. For example, the introduction of a loop at the T-1 turn might, for some reason, be especially destabilizing. In this context it might be noted that neither mutant showed looping out at T-1. Rather, it occurs in both cases at site T-3. Alternatively, the resulting loss of the His31-Asp70 salt bridge might not be crucially important. As noted above, a tyrosine may be a favorable substitute for a histidine in the right circumstances. In yet another scenario, the looping out of the sequence at T-3 might allow an especially favorable structure to form that could offset the other destabilizing changes. Against this, however, the loop structure at T-3 in the L30c mutant does not appear to be especially rigid and requires buttressing crystal contacts to be visualized in the crystal structure. Also, prior studies suggest that sequences within beta-sheet strands (at least for T4 lysozyme) do not have a strong intrinsic folding propensity (Najbar et al. 1997; Sagermann and Matthews 2002; He et al. 2004).

At this point, the crystal structures do not offer any clear insights on which interactions define the beta-sheet topology. The folding of L30c proves that even a strong salt-bridge interaction can be sacrificed to accommodate the new insert while still maintaining the native-like fold. In light of the fact that the amino acid sequence in this vicinity does not have a high intrinsic propensity to form either the turns or the beta-sheet strands, one needs to question whether a hierarchical folding mechanism may be responsible for the observed topology. The sequence Gly28-Ile-Gly30 does not have a strong propensity to form turns. These three residues are, however, buried and make multiple interactions with residues within the C-terminal domain (including helix C). If the C-terminal domain folds first (Lu and Dahlquist 1992; Gassner et al. 1999), then the interactions of Gly28-Ile-Gly30 with this domain may be especially important in directing the folding of the beta-sheet region within the N-terminal domain. Such a mechanism would also explain the acceptance of a non-native sequence in the folding of the beta-sheet, notwithstanding a loss of stability.

Alternative splicing is frequently observed in loop sequences of proteins. It is estimated that approximately one-third to one-half of all human genes become alternatively spliced (Kondrashov and Koonin 2003). Short sequences or entire domains can be edited into a protein sequence in this way. Thus far, only a few naturally occurring insertions into protein structures have been characterized structurally. Most recently, the structures of the human pyrophosphorylase AGX1 and AGX2 illustrate how naturally occurring splice inserts are accommodated in the protein. Whereas the sequences of most inserts usually loop out at the insertion site, in this case an alternatively spliced sequence of nine residues displaces Strand 4 within the beta-sheet. The displaced strand adopts an {alpha}-helical conformation instead (Garcia et al. 2004). As the insert and the sequence of Strand 4 compete for the same place in the architecture of the protein, it is most interesting that the insert structure appears to have the higher affinity.

Another example of a dramatic structural reorganization of beta-sheet structure is observed in SERPINS. Upon proteolytic cleavage of a surface loop, a nine–amino acid sequence segment is liberated and inserts itself into the center of a beta-sheet (Carrell et al. 1994). Even though the sheet topology has changed dramatically, the overall structure of the protein changes very little.

The mutants described here illustrate the ability of a protein to accommodate such insertions. They also illustrate how duplications of secondary structures could accelerate protein evolution. It is generally accepted that insertions in protein sequences are accommodated by changes in surface loops. Our results are consistent with this, but also show how an insertion can be made at one site but result in substantial changes in structure in a loop that is remote from the site of the insertion.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Mutagenesis
All the insertions were introduced into a cysteine-free, pseudo-wild-type T4 lysozyme gene (WT*) (Matsumara and Matthews 1989). Insertions were carried out using standard, two-stage PCR. Using the Escherichia coli codon frequency list, the codons of the inserted DNA segment were chosen to mismatch the codons of the parent sequence. For each of the listed mutants, the insertion carrying primers are shown. The outermost primers that flank the T4 lysozyme gene are: 5'-CCCTGTTGACAATTAATCATCGG-3'; 5'-GGCGTATCACGAGGCCC-3'. For mutant L30c, the amino acid sequence Tyr-Thr-Ile-Gly-Ile-Gly was duplicated in tandem with the primers: 5'-GACACAGAAGGCTATTATACCATCGGTATTGGCTACACTATTGGCATCGG-3'; 5'-CGGATGCCAATAGTGTAGCCAATACCGTAGGTATAATAGCCTTCTGTGTC-3'. The sequence of mutant L31d, Gly-Ile-Gly-His-Leu-Leu, was inserted using the primers: 5'-GACACAGAAGGCTATTACACTATTGGTATTGGCCACCTTCTGGGCATCGGTCATTTGCTTACAAAAAG-3'; 5'-CTTTTTGTAAGCAAATGACCGATGCCCAGAAGGTGGCCAATACCAATAGTCTAATAGCCTTCTGTGTC-3'.

The resulting genes were cloned into the expression vector pHS1403 using the BamH1 and HindIII restriction sites, and subsequently transformed into RR1 cells. Expression and purification of the mutant proteins were performed as described (Poteete et al. 1991). The sequences of the resulting clones were verified by nucleotide sequencing, test expression of the protein, and mass spectroscopy analysis of the purified proteins. Solubility, monodispersity, and activity were confirmed as described (Heinz et al. 1993).

Crystallography
Both mutant proteins were dialyzed against 50 mM Tris-HCl (pH 7.5) and 100 mM NaCl and concentrated to ~20 mg/mL. As judged by dynamic light-scattering, both proteins appeared to be monodisperse. Crystallization conditions were identified by scanning 48 conditions of the Hampton Crystal Screen 2. A variety of different crystallization conditions resulted in protein crystals. Mutant L30c crystallized optimally out of the precipitate using 15% polyethylene glycol 4000, Tris-HCl (pH 7.8), and 50 mM ammonium sulfate within ~2 wk. The crystals grew to thin plates of ~0.3 x 0.2 x 0.02 mm. Mutant L31d crystallized in 20% polyethylene glycol 3400, 50 mM Tris-HCl (pH 7.5), and 50 mM ammonium sulfate. First crystals could be observed within 10 h. Suitable crystals grew within 1 wk to needles of ~0.02 x 0.01 x 0.4 mm. Even though the crystallization conditions are similar, the two mutants crystallized in different space groups (Table 1). Neither is isomorphous with crystals of WT*.


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  		Table 1. X-ray data collection and refinement statistics

 
Data sets for mutant L31d were collected in-house on a Rigaku (Tokyo) R-AXISIIc image plate detector. Data sets were integrated with XDS (Kabsch 1988, 1993). Synchrotron data sets for mutant L30c were obtained at the Advanced Light Source (ALS BL5.0.2) and at the Stanford Synchrotron Radiation Laboratory (SSRL BL9–1). Data integration and reduction were performed using the HKL2000 (Otwinowski and Minor 1997) and the MOSFLM/SCALA (Leslie 1992; CCP4 1994) suite of programs, respectively. All data collection was performed on flash-frozen crystals.

For all mutants, the structures were determined by molecular replacement with the program AMoRe (CCP4 1994; Navaza 1994) using either wild-type T4 lysozyme or mutants with different hinge bending angles (Zhang et al. 1995) as search models. In addition, the C-terminal domain only was also tried as a search model to further confirm the positions of the molecules in the asymmetric unit. The best solutions were refined using CNS v1.1 (Brünger et al. 1998), TNT (Tronrud et al. 1987; Tronrud 1996), and REFMAC (CCP4 1994). Model building was performed in O (Jones et al. 1991). The correctness of the structures was further checked by systematic calculations of simulated-annealing-omit maps. For all mutants, several independent data sets from different crystals were obtained and independently refined (data not shown). The refinement of all mutant structures was performed without the application of any local symmetry operators to avoid averaging of structures in the asymmetric unit.

Stability measurements
The stability of each mutant protein was monitored by observing the change in circular dichroism as a function of temperature. Experimental procedures were as described previously (Eriksson et al. 1993). At pH 5.4, solutions of mutant L30c (0.0004–0.0030 mg/mL) in 0.1 M NaCl, 0.01 M NaOAc had a melting temperature of 55.5 ± 0.5°C. Under similar conditions, WT* melts at 65.5°C. Using the relationship of Becktel and Schellman (1987), this corresponds to a loss in stability of ~3.7 kcal/mol. When mutant L31d was analyzed under similar conditions it appeared to unfold at ~57.5°C, but the unfolding was not reversible. This means that a reliable determination of the free energy is not possible, but it does appear that the mutation results in a loss of stability of ~3 kcal/mol.


   Footnotes
 
1 Present address: Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA. Back

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.052018006.

Reprint requests to: Brian Matthews, Institute of Molecular Biology, Howard Hughes Medical Institute, and Department of Physics, 1229 University of Oregon, Eugene, OR 97403-1229, USA; e-mail: brian{at}uoxray.uoregon.edu; fax: (541) 346-5870.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.052018006.


   Acknowledgments
 
We thank the staff at ALS, beamline 5.0.2, for their expert help with data collection. This work was supported in part by NIH grant GM21967 to B.W.M.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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Eriksson A.E., Baase W.A., Matthews B.W. 1993. Similar hydrophobic replacements of Leu99 and Phe153 within the core of T4 lysozyme have different structural and thermodynamic consequences J. Mol. Biol. 229: 747–769.[CrossRef][Medline]

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