Extended conformations of polypeptides and proteins in urea and guanidine hydrochloride

Tiffany, M. Lois; Krimm, Samuel

doi:10.1002/bip.1973.360120310

Cited by 131 publications

(102 citation statements)

References 38 publications

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“…Further, it was destroyed by nonaqueous solvent, by removing solvents, and by fragmentation with trypsin and heating. On the other hand, the addition of 6 M urea did not appear to affect the 227 nm band, an observation that could be consistent with the presence of this conformation (35). A stumbling block to this interpretation is that the position of the band, 227 nm, was about 10 nm higher than that usually reported for charged polypeptides.…”

Section: Resultssupporting

confidence: 72%

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The Secondary Structure of Human Hageman Factor (Factor XII) and its Alteration by Activating Agents

McMillin¹,

Saito²,

Ratnoff³

et al. 1974

J. Clin. Invest.

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A B S T R A C T Hageman factor (factor XII) is activated by exposure to surfaces such as glass or by solutions of certain compounds, notably ellagic acid. Changes in the structure of Hageman factor accompanying activation have been examined in this study by circular dichroism spectroscopy. The spectrum of unactivated Hageman factor in aqueous solutions suggests that its conformation is mainly aperiodic. Various perturbants altered the conformation of Hageman factor in differing ways, demonstrating the sensitivity of Hageman factor to its environment.After activation of Hageman factor with solutions of ellagic acid, a negative trough appeared in the region of the circular dichroism spectrum commonly assigned to tyrosine residues, along with other minor changes in the peptide spectral region. Some of these changes are similar to changes that occurred upon partial neutralization of the basic residues at alkali pH. Activation of Hageman factor by adsorption to quartz surfaces (in an aqueous environment) also produced changes similar to those in the ellagic acid-activated Hageman factor, including the negative ellipticity in the tyrosine region.These observations suggest that the activation process may be related to a change in status of some of the basic amino acid residues, coupled with a specific change in the environment of some tyrosine residues. The importance of these changes during the activation process remains to be determined. The

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Section: Resultssupporting

confidence: 72%

“…The third aspect of protein structure analysis by CD spectroscopy is that many proteins including bovine HF (39) (35,36). These conditions produce only weak negative optical activity above about 215 nm and a weak negative CD trough around 195 nm.…”

Section: Introductionmentioning

confidence: 99%

The Secondary Structure of Human Hageman Factor (Factor XII) and its Alteration by Activating Agents

McMillin¹,

Saito²,

Ratnoff³

et al. 1974

J. Clin. Invest.

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“…The finding that guanidinium promotes polyproline II (PPII) structure was originally taken to indicate that guanidinium H-bonds to the peptide group (38). In light of our finding that such H-bonding is absent, it is plausible that the mode of interaction that leads to the maximally exposed PPII main chain conformation is a space-demanding stacking of guanidinium against the peptide group.…”

Section: Discussionmentioning

confidence: 90%

“…Guanidinium could H-bond to the peptide carbonyl and thus block the carbonyl protonation step of acid-catalyzed HX, but this does not occur beyond the level of random encounter, again ruling out Hbonding. This is surprising because guanidinium is thought to H-bond to the peptide carbonyl (10,11,(36)(37)(38). On the other hand, the absence of guanidinium to peptide H-bonding is consistent with the behavior of guanidinium in solution.…”

Section: Discussionmentioning

confidence: 98%

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Lim

Rösgen

Englander

2009

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

360

370

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The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid-and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea. Results for guanidinium, however, are contrary to the expectation that it might H-bond. Evidently, urea and guanidinium, although structurally similar, denature proteins by different mechanisms.denaturation ͉ hydrogen exchange ͉ osmolyte ͉ solvation B ecause of its fundamental importance, the study of protein molecular stability has engaged the attention of protein chemists for the last 100 years (1, 2). Experimental and theoretical investigations of protein stability have often focused on small-molecule osmolytes that can be used to modulate stability in vitro (3-6) and are used by virtually all organisms to counter biochemical stress in vivo (7). The mechanism of osmolyte action continues to be controversial. Opposing positions favor either a direct interaction between protein and osmolyte (8-11), such as hydrogen bonding, or an indirect effect mediated by the alteration of water structure (12, 13), or a mixture of both (14)(15)(16)(17)(18)(19). It has been difficult to distinguish between these direct and indirect models because the osmolyte-protein interaction is so weak.It is a thermodynamic truism (20-22) that the concentration of destabilizing osmolytes must be enriched relative to water molecules in the vicinity of the protein surface that becomes newly exposed upon unfolding (preferential interaction). In a thermodynamic sense, denaturing osmolytes ''bind'' selectively to the increased surface of unfolded proteins and thus bias the folded/ unfolded equilibrium toward denaturation. Similarly, stabilizing osmolytes must be preferentially excluded from the protein surface. This is true independently of the physical mechanism of the ''binding'' or ''antibinding'' interaction. Thermodynamically based studies directed at the binding/antibinding problem have been designed to measure the transfer free energy of the various component groups of proteins between water and osmolyte solutions. Results show that the main-chain peptide group makes the largest contribution to the energetics, accounting for Ϸ80% of the effect of urea and of strong stabilizers such as trimethyl amine N-oxide (TMAO) (23). Similar determinations for guanidinium are not yet available, but it seems clear that guanidinium also favorably interacts with the peptide group (8,11,24).To search for the mechanistic basis of thermodynamic destabilization due to urea and guanidinium, we tested the possibility that they exert their denaturing effect through peptide group hydrogen bonding. This can be done experimentally by the straightforward measurement of acid-and base-catalyzed hydrogen exchange (HX) in a small-molecule peptide model. Osmolyte that is H-bonded to the peptide NH ...

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“…4A) displays an isodichroic point at 215 nm, indicating an equilibrium of two major populations of conformations. Another unique characteristic of PII is its response to urea and guanidinium chloride treatment (23). These chaotropic agents that commonly cause unfolding and loss of most secondary and tertiary structures in fact increase the helical content of PPII (23).…”

Section: Resultsmentioning

confidence: 99%

Modular Motif, Structural Folds and Affinity Profiles of the PEVK Segment of Human Fetal Skeletal Muscle Titin

Gutierrez-Cruz

Heerden

Wang

2001

Journal of Biological Chemistry

109

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The extension of the PEVK segment of the giant elastic protein titin is a key event in the elastic response of striated muscle to passive stretch. PEVK behaves mechanically as an entropic spring and is thought to be a random coil. cDNA sequencing of human fetal skeletal PEVK reveals a modular motif with tandem repeats of modules averaging 28 residues and with superrepeats of seven modules. Conformational studies of bacterially expressed 53-kDa fragment (TP1) by circular dichroism suggest that this soluble protein contains substantial polyproline II (PPII) type left-handed helices. Urea and thermal titrations cause gradual and reversible decrease in PPII content. The absence of sharp melting in urea and thermal titrations suggests that there is no long range cooperativity among the PPII helices. Studies with solid phase and surface plasmon resonance assays indicate that TP1 interacts with actin and some but not all cloned nebulin fragments with high affinity. Interestingly, Ca 2؉ /calmodulin and Ca 2؉ /S100 abolish nebulin/PEVK interaction. We suggest that in aqueous solution, PEVK is an open and flexible chain of relatively stable structural folds of the polyproline II type. PEVK region of titin may be involved in interfilament association with thin filaments in a calcium/calmodulinsensitive manner. This adhesion may modulate titin extensibility and elasticity.The monumental sequencing work of Labeit and Kolmerer (1) has revealed the complete domain organization of the giant elastic protein titin. The bulk of cardiac titin consists of predominately two types of sequence motifs: immunoglobulin (Ig) and fibronectin arranged in three levels of motifs (repeats, superrepeats, and segments). In addition to these well characterized domains, a novel motif consisting of mainly four amino acid residues, Pro, Glu, Val, and Lys, is discovered in the elastic I band region of titin. The length of this PEVK segment varies among muscles, ranging from 183 residues in the human cardiac titin to 2174 residues in human soleus skeletal muscle. Differential splicing of the titin transcripts in the PEVK region as well as in an adjacent tandem Ig segment near the A band produce these size isoforms of titin (1, 2). Since the selective expression of titin size isoforms imparts distinct elasticity to skeletal and cardiac muscles, with the muscle expressing longer titin being more compliant (3), the observed length variation of PEVK and tandem Igs in titin isoforms immediately suggests the possibility that either or both segments may be the elastic elements. Labeit and Kolmerer (1) speculated that the PEVK region, with a predicted nonfolded polypeptide, is the key elastic element of titin. The concept of reversible unfolding and folding of Ig domains was considered unlikely, on the ground of the thermodynamic stability of Ig domains (4, 5).Recent works on the elasticity of single titin molecules (6), single myofibrils (7), and single fibers (8, 9) revealed that, stretched modestly, sarcomere elasticity can be explained by the straightening...

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Extended conformations of polypeptides and proteins in urea and guanidine hydrochloride

Cited by 131 publications

References 38 publications

The Secondary Structure of Human Hageman Factor (Factor XII) and its Alteration by Activating Agents

The Secondary Structure of Human Hageman Factor (Factor XII) and its Alteration by Activating Agents

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Modular Motif, Structural Folds and Affinity Profiles of the PEVK Segment of Human Fetal Skeletal Muscle Titin

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