Proteins as random coils.  III.  Optical rotatory dispersion in 6M guanidine hydrochloride

Tanford, Charles; Kawahara, Kazuo; Lapanje, Savo; Hooker, Thomas M.; Zarlengo, Mario H.; Salahuddin, A; Aune, Kirk C.; Takagi, Toshio

doi:10.1021/ja00995a034

Cited by 123 publications

(58 citation statements)

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“…2). Superimposing 3 J HN␣ values for each site in the chain reveals a common behavior in their temperature dependence: a monotonic increase in coupling constant between 2 and 56°C (Fig. 3).…”

Section: Resultsmentioning

confidence: 89%

“…Our primary objective is to measure the 3 J HN␣ coupling constant that is related directly to the angle (20,21). The one-dimensional spectrum must be well resolved in the NH region to get accurate 3 The coupling constants for different residues determined in this way change markedly with temperature ( Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…T anford's pioneering experiments on denatured proteins in 6 M guanidinium chloride (GdmCl) (1)(2)(3)(4)(5) were interpreted by using the random coil model, and they anchored a widespread belief that denatured proteins are structureless chains. Tanford emphasized that 6 M GdmCl is required to eliminate all residual structure, which was detected by optical rotatory dispersion in heat-denatured proteins (6).…”

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confidence: 99%

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Polyproline II structure in a sequence of seven alanine residues

Shi

Olson

Rose

et al. 2002

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

507

106

682

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A sequence of seven alanine residues-too short to form an ␣-helix and whose side chains do not interact with each other-is a particularly simple model for testing the common description of denatured proteins as structureless random coils. The 3 JHN␣ coupling constants of individual alanine residues have been measured from 2 to 56°C by using isotopically labeled samples. The results display a thermal transition between different backbone conformations, which is confirmed by CD spectra. The NMR results suggest that polyproline II is the dominant conformation at 2°C and the content of ␤ strand is increased by approximately 10% at 55°C relative to that at 2°C. The polyproline II conformation is consistent with recent studies of short alanine peptides, including structure prediction by ab initio quantum mechanics and solution structures for both a blocked alanine dipeptide and an alanine tripeptide. CD and other optical spectroscopies have found structure in longer ''random coil'' peptides and have implicated polyproline II, which is a major backbone conformation in residues within loop regions of protein structures. Our result suggests that the backbone conformational entropy in alanine peptides is considerably smaller than estimated by the random coil model. New thermodynamic data confirm this suggestion: the entropy loss on alanine helix formation is only 2.2 entropy units per residue. T anford's pioneering experiments on denatured proteins in 6 M guanidinium chloride (GdmCl) (1-5) were interpreted by using the random coil model, and they anchored a widespread belief that denatured proteins are structureless chains. Tanford emphasized that 6 M GdmCl is required to eliminate all residual structure, which was detected by optical rotatory dispersion in heat-denatured proteins (6). The random coil model has been applied to modern NMR studies of backbone conformation in denatured proteins (7,8) by assuming that the backbone conformations found in protein structures, including or excluding regions of regular secondary structure, are represented with the same frequencies in denatured proteins. One might suppose, however, that the energy differences between major backbone conformations are sufficiently large to favor one conformation over others, at least in a homopeptide.We address this question by using NMR to investigate the backbone conformation as a function of temperature for a sequence of seven alanine residues in a peptide solubilized in water by two basic residues at either end of the alanine sequence. This peptide presents an appealing model system for a structureless denatured protein. It is too short to form any detectable ␣-helix in water via peptide hydrogen bonds; moreover, the OCH 3 side chain is too short to form nonpolar clusters and too inert to form other side chain interactions. To characterize the backbone conformations of this peptide, we measure system properties that are related directly to either the or the backbone angles. It is possible today to resolve and assign most backbone resonances of de...

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Polyproline II structure in a sequence of seven alanine residues

Shi

Olson

Rose

et al. 2002

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

507

106

682

View full text Add to dashboard Cite

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“…It is a daunting task to attempt isolation and analysis of denatured conformational isomers, not only because of the exceedingly large number of isomers that may exist, but also because of their instability and rapid interconversion. Without isolation of conformational isomers, the structural property of a denatured protein has been typically concluded from the measurement of the collective isomers using various spectroscopic techniques (47)(48)(49)(50)(51). This has been the case for the analysis of most denatured proteins, including ␣-LA.…”

Section: Discussionmentioning

confidence: 99%

The Structure of Denatured α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

Chang

2001

Journal of Biological Chemistry

100

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The structure of denatured ␣-lactalbumin (␣-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, ␣-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured ␣-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured ␣-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of ␣-LA. Most importantly, unique isomers of partially unfolded ␣-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic ␣-helical domain, generating a predominant isomer containing two native disulfide bonds at the ␤-sheet domain and two scrambled disulfide bonds at the ␣-helical region. Thermal denaturation selectively unfolds the ␤-sheet domain of ␣-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of ␣-LA. ␣-LA1 represents one of the most extensively investigated models for understanding the mechanism of protein stability, folding, and unfolding. Under a variety of conditions, ␣-LA adopts a partially structured conformation termed as "molten globule" (1-9), which has been observed along both the unfolding (1, 2, 5, 10 -12) and folding pathways (13-18) of ␣-LA and is considered one of the best characterized folding/unfolding intermediates of globular proteins. At low pH (1,19,20), elevated temperature (19, 21), or mild concentration of denaturant (e.g. GdmCl; Refs. 22, 23), the native ␣-LA unfolds to the state of a molten globule. During the refolding, the fully denatured ␣-LA undergoes a rapid collapse to the state of molten globule (13,14,18) as intermediate, which is followed by consolidation and search of side chain interactions to attain the native protein (14). The structure of ␣-LA molten globule is characterized by a high degree of native-like secondary structure and a fluctuated tertiary fold (1)(2)(3)(4)19). It is stabilized mainly by the core hydrophobic amino acids within the ␣-helical domain (24 -28) and may form even in the absence of its four disulfide bonds (15). The structure of the molten globule of ␣-LA is also highly heterogeneous (3, 27, 29). Like most denatured or partially denatured state of proteins (30, 31), it comprises a large number of conformational isomers. So far, characterization of the structure of denatured ␣-LA and the molten globule state of ␣-LA have been achieved by measuring the average property of these collective isomers using a wide range of spectroscopic and physiochemical methods, including circular dichroism (1, 2, ...

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“…Flory predicted the exponent to be 0.6 and later a more accurate renormalization calculation obtained ν=0.588 [189]. Tanford et al first confirmed this random coil scaling behavior for denatured proteins [190]. Using intrinsic viscosity measurements for 12 proteins denatured by 5-6 M GuHCl, the authors obtained a scaling exponent ν=0.67±0.09.…”

Section: Unfolded Protein Statesmentioning

confidence: 95%

Protein folding: Then and now

Chen

Ding

Nie

et al. 2008

Archives of Biochemistry and Biophysics

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Over the past three decades the protein folding field has undergone monumental changes. Originally a purely academic question, how a protein folds has now become vital in understanding diseases and our abilities to rationally manipulate cellular life by engineering protein folding pathways. We review and contrast past and recent developments in the protein folding field. Specifically, we discuss the progress in our understanding of protein folding thermodynamics and kinetics, the properties of evasive intermediates, and unfolded states. We also discuss how some abnormalities in protein folding lead to protein aggregation and human diseases.

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Proteins as random coils. III. Optical rotatory dispersion in 6M guanidine hydrochloride

Cited by 123 publications

References 0 publications

Polyproline II structure in a sequence of seven alanine residues

Polyproline II structure in a sequence of seven alanine residues

The Structure of Denatured α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

Protein folding: Then and now

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